Removal of serine proteases by treatment with finely divided silicon dioxide

ABSTRACT

The present invention provides novel methods for reducing the serine protease and/or serine protease zymogen content of a plasma-derived protein composition. Also provided are methods for manufacturing plasma-derived protein compositions having reduced serine protease and\or serine protease zymogen content. Among yet other aspects, the present invention provides aqueous and lyophilized compositions of plasma-derived proteins having reduced serine protease and/or serine protease zymogen content. Yet other aspects include methods for treating, managing, and/or preventing a disease comprising the administration of a plasma-derived protein composition having a reduced serine protease or serine protease zymogen content.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.13/117,028, filed May 26, 2011, which is a continuation of U.S. patentapplication Ser. No. 12/842,944, filed Jul. 23, 2010; U.S. patentapplication Ser. No. 13/117,028, filed May 26, 2011, is a continuationof U.S. patent application Ser. No. 12/789,365, filed May 27, 2010, andclaims priority to AU Patent Application No. 2010202125, filed May 26,2010 (issued as Australian Patent No. 2010202125), the disclosures ofwhich are hereby incorporated herein by reference in their entiretiesfor all purposes.

BACKGROUND OF THE INVENTION

Plasma-derived blood products are used to treat not only a variety ofblood disorders, but diseases of other origin. For example, immuneglobulin (IgG) products from human plasma were first used in 1952 totreat immune deficiency. Since then, IgG preparations have foundwidespread use in at least three main categories of medical conditions:(1) immune deficiencies such as X-linked agammaglobulinemia,hypogammaglobulinemia (primary immune deficiencies), and acquiredcompromised immunity conditions (secondary immune deficiencies),featuring low antibody levels; (2) inflammatory and autoimmune diseases;and (3) acute infections.

Likewise, Factor H has been implicated as a potential therapeutic agentfor several human disease states, including age-related maculardegeneration (AMD), hemolytic uremic syndrome (aHUS) andmembranoproliferative glomerulonephritis (MPGN). Specifically, a causalrelationship between the single nucleotide polymorphism (SNP) incomplement control protein (CCP) module 7 of Factor H and age-relatedmacular degeneration (AMD) has been characterized.

Studies have shown correlations between decreased plasma levels ofInter-alpha-Inhibitor proteins (IaIp) and mortality in patients withsevere sepsis (Lim et al., J Infect Dis. (2003) September 15;188(6):919-26 and Opal et al., Crit Care Med. (2007) February;35(2):387-92). Furthermore, several studies have shown that theadministration of IaIp reduces mortality associated with sepsis andseptic shock (Jourdain et al., Am J Respir Crit Care Med. (1997)December; 156(6):1825-33; Yang et al., Crit Care Med. (2002) March;30(3):617-22; Lim et al., J Infect Dis. (2003) September 15;188(6):919-26; and Wu et al., Crit Care Med. (2004) August;32(8):1747-52; the disclosures of which are incorporated by referenceherein in their entireties for all purposes).

Various safety precautions must be taken into consideration whenmanufacturing and formulating plasma-derived biologic therapies. Theseinclude methods for removing and/or inactivating blood borne pathogens(e.g., viral and bacterial pathogens), anticomplement activity, andother unwanted contaminants arising from the use of donated plasma.Studies have suggested that administration of high levels of amidolyticactivity may result in unwanted thromboembolic events (Wolberg A S etal., Coagulation factor XI is a contaminant in intravenousimmunoglobulin preparations. Am J Hematol 2000; 65:30-34; and Alving B Met al., Contact-activated factors: contaminants of immunoglobulinspreparations with coagulant and vasoactive properties. J Lab Clin Med1980; 96:334-346; the disclosures of which are hereby incorporated byreference in their entireties for all purposes). Highlighting thisconcern was the recent voluntary withdrawal of Octagam® (Octapharma) inthe US and suspension of marketing authorization for Octagam® andoctagam 10% by the European Commission following increased reports ofthromboembolic events. It is likely that the increased thrombolic eventswere caused by high levels of amidolytic activity in the biologic,caused by serine protease and serine protease zymogen impurities, suchas Factor XI, Factor XIa, Factor XII and Factor XIIa (FDA Notice:Voluntary Market Withdrawal—Sep. 23, 2010 Octagam [Immune GlobulinIntravenous (Human)] 5% Liquid Preparation; Octagam 50 mg/ml, solutionpour perfusion—Octapharma France—Mise en quarantaine de tous les lots,published online Sep. 9, 2010 by the AFSSAPS; and Questions and answerson the suspension of the marketing authorisations for Octagam (humannormal immunoglobulin 5% and 10%), published online Sep. 23, 2010 by theEuropean Medicines Agency).

Dedicated serine proteases, generically known as coagulation factors,are integral components of both the contact activation and tissue factorpathways of the coagulation cascade. Upon a stimulus of the coagulationpathways, serine protease zymogens, which are inactive enzymeprecursors, become activated proteases that catalyze the activation ofthe next protease zymogen, resulting in an activation cascade. Thiscoagulation cascade culminates in the activation of Thrombin (FactorIIa) and Factor XIIIa, which function to convert Fibrinogen (Factor I)into Fibrin (Factor Ia) and cross-link fibrin to form a fibrin clot,respectively.

The contact activation pathway, also known as the intrinsic coagulationpathway, begins with the activation of Kallikrein and Factor XIIa(FXIIa) from Prekallikrein and Factor XII, respectively. The activatedserine protease FXIIa cleaves Factor XI (FXI), converting the zymogeninto Factor XIa (FXIa), an active serine protease which participates inthe subsequent activation of Factor Xa (FXa).

Due to rising concerns over the presence of serine protease and serineprotease zymogens in plasma-derived protein compositions, there remainsa need in the art for methods for reducing the levels of thesecontaminants, and particularly FXI, FXIa, FXII, and FXIIa. The presentinvention fulfils these and other needs by providing such methods andplasma-derived protein compositions with reduced levels of serineprotease and serine protease zymogen.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention is based on the surprising findingthat serine proteases and serine protease zymogens, and specifically,FXI, FXIa, FXII, and FXIIa, can be removed from plasma-derived proteincompositions by treatment with finely divided silicon dioxide (SiO₂). Inthis fashion the present invention provides methods for reducing theserine protease activity, serine protease content, and serine proteasezymogen content of plasma-derived protein compositions. Also providedare therapeutic plasma-derived protein compositions having reducedserine protease activity, serine protease content, and serine proteasezymogen content, as well as methods for treating or preventing diseaseby the administration of the same.

In a first aspect, the present invention provides a method for reducingthe amount of a serine protease or a serine protease zymogen in aplasma-derived target protein composition, the method comprising thesteps of: (a) contacting the composition with finely divided silicondioxide (SiO₂) under conditions suitable to bind at least one serineprotease or serine protease zymogen; and (b) separating the SiO₂ fromthe composition to remove the bound serine protease. In a preferredembodiment, the serine protease or serine protease zymogen is Factor XIa(FXIa), Factor XIIa (FXIIa), Factor XI (FXI), or Factor XII (FXII).

In certain embodiments, the method described above further comprises thestep of performing a first target protein enrichment step to form afirst enriched composition, prior to contacting the composition withfinely divided silicon dioxide (SiO₂). In one embodiment, the firsttarget protein enrichment step is a protein precipitation step. In aspecific embodiment, the protein precipitation step is an alcoholfractionation step. In another embodiment, the first target proteinenrichment step is an ultrafiltration/diafiltration step.

In other embodiments of the methods described above, the method furthercomprises the step of performing a second target protein enrichment stepprior to contacting the enriched composition with finely divided silicondioxide (SiO₂). In one embodiment, the second target protein enrichmentstep is a protein precipitation step. In a specific embodiment, theprotein precipitation step is an alcohol fractionation step. In anotherembodiment, the second target protein enrichment step is anultrafiltration/diafiltration step. In yet another embodiment, thesecond target protein enrichment step is a chromatographic enrichmentstep.

In other embodiments of the methods described above, the method furthercomprises the step of performing a third target protein enrichment stepafter contacting the composition with finely divided silicon dioxide(SiO₂). In one embodiment, the third target protein enrichment step is aprotein precipitation step. In a specific embodiment, the proteinprecipitation step is an alcohol fractionation step. In anotherembodiment, the third target protein enrichment step is anultrafiltration/diafiltration step. In yet another embodiment, the thirdtarget protein enrichment step is a chromatographic enrichment step.

In certain embodiments of the methods described above, thechromatographic enrichment step comprises the sub-steps of: (i)contacting the plasma-derived target protein composition with achromatographic resin under conditions suitable to bind theplasma-derived target protein; and (ii) eluting the plasma-derivedtarget protein from the chromatographic resin. In a specific embodiment,the impurity does not bind to the chromatographic resin in sub-step (i).In another specific embodiment, the impurity binds to thechromatographic resin in sub-step (i), but is not eluted from thechromatographic resin in sub-step (ii).

In other certain embodiments of the methods described above, thechromatographic enrichment step comprises the sub-steps of: (i)contacting the first enriched plasma-derived target protein compositionwith a chromatographic resin under conditions suitable to bind at leastone impurity; and (ii) separating the resin from the plasma-derivedprotein composition, wherein the plasma-derived target protein does notbind to the chromatographic resin in sub-step (i).

In certain embodiments of the methods comprising a chromatographicenrichment step described above, the chromatographic resin is selectedfrom the group consisting of an anion exchange resin, a cation exchangeresin, a hydrophobic interaction resin, a mixed mode resin, ahydroxyapatite resin, a ligand affinity resin, an immunoaffinity resin,and a size exclusion resin.

In other certain embodiments of the methods comprising a chromatographicenrichment step described above, the chromatographic enrichment stepcomprises separating at least one impurity from the target protein bysize and/or shape using size exclusion chromatography.

In certain embodiments of the methods described above, theplasma-derived target protein is selected from an immunoglobulin (Ig),albumin, alpha-1-antitrypsin (A1PI), butyrylcholinesterase, a protein ofthe complement system, and an inter-alpha-trypsin inhibitor (IαI). In aspecific embodiment, the protein of the complement system is selectedfrom the group consisting of Factor H (FH), Factor D, complement proteinC3, and C4 binding protein.

In yet another embodiment of the methods described above, theplasma-derived target protein composition is a manufacturingintermediate.

In a second aspect, the present invention provides a method forpreparing a plasma-derived Factor H composition having a reduced amountof a serine protease or a serine protease zymogen, the method comprisingthe steps of: (a) contacting a composition containing Factor H and atleast one serine protease or serine protease zymogen with finely dividedsilicon dioxide (SiO₂) under conditions suitable to bind the Factor Hand at least one serine protease or serine protease zymogen; (b)separating the SiO₂ from the composition; (c) eluting the serineprotease or serine protease zymogen from the SiO₂ under a solutioncondition in which the Factor H remains bound; and (d) eluting theFactor H from the SiO₂.

In certain embodiment of the methods described above, the solutioncondition under which the serine protease or serine protease zymogen iseluted from the SiO₂ and the Factor H remains bound comprises a pHgreater than about 6.0. In another embodiment, the solution conditionunder which the serine protease or serine protease zymogen is elutedfrom the SiO₂ and the Factor H remains bound comprises a pH greater thanabout 6.5. In another embodiment, the solution condition under which theserine protease or serine protease zymogen is eluted from the SiO₂ andthe Factor H remains bound comprises a pH greater than about 7.0. In yetanother embodiment, the solution condition under which the serineprotease or serine protease zymogen is eluted from the SiO₂ and theFactor H remains bound comprises a pH of at least about 7.5.

In certain embodiment of the methods described above, the solutioncondition comprises a pH of no more than 11.0. In another embodiment,the solution condition comprises a pH of no more than 10.0. In anotherembodiment, the solution condition comprises a pH of no more than 9.0.

In certain embodiment of the methods described above, the solutioncondition under which the serine protease or serine protease zymogen iseluted from the SiO₂ and the Factor H remains bound comprises aconductivity of greater than about 10 mS/cm. In another embodiment ofthe methods described above, the solution condition under which theserine protease or serine protease zymogen is eluted from the SiO₂ andthe Factor H remains bound comprises a conductivity of greater thanabout 20 mS/cm. In another embodiment of the methods described above,the solution condition under which the serine protease or serineprotease zymogen is eluted from the SiO₂ and the Factor H remains boundcomprises a conductivity of between about 10 mS/cm and about 50 mS/cm.In yet another embodiment of the methods described above, the solutioncondition under which the serine protease or serine protease zymogen iseluted from the SiO₂ and the Factor H remains bound comprises aconductivity between about 20 mS/cm and about 50 mS/cm.

In a third aspect, the present invention provides a method for preparinga Factor H composition having a reduced amount of a serine protease or aserine protease zymogen, the method comprising the steps of: (a)contacting a composition containing Factor H and at least one serineprotease or serine protease zymogen with finely divided silicon dioxide(SiO₂) under conditions suitable to bind the Factor H and at least oneserine protease; (b) separating the SiO₂ from the composition; and (c)eluting the Factor H from the SiO₂ under conditions in which the serineprotease or serine protease zymogen remains bound to the SiO₂.

In certain embodiment of the methods described above, the solutioncondition under which the Factor H is eluted from the SiO₂ and theserine protease or serine protease zymogen remains bound comprises a pHgreater than about 6.0. In another embodiment, the solution conditionunder which the Factor H is eluted from the SiO₂ and the serine proteaseor serine protease zymogen remains bound comprises a pH greater thanabout 6.5. In another embodiment, the solution condition under which theFactor H is eluted from the SiO₂ and the serine protease or serineprotease zymogen remains bound comprises a pH greater than about 7.0. Inyet another embodiment, the solution condition under which the Factor His eluted from the SiO₂ and the serine protease or serine proteasezymogen remains bound comprises a pH of at least about 7.5.

In certain embodiment of the methods described above, the solutioncondition comprises a pH of no more than 11.0. In another embodiment,the solution condition comprises a pH of no more than 10.0. In anotherembodiment, the solution condition comprises a pH of no more than 9.0.

In certain embodiment of the methods described above, the solutioncondition under which the Factor H is eluted from the SiO₂ and theserine protease or serine protease zymogen remains bound comprises aconductivity of less than about 20 mS/cm. In another embodiment, thesolution condition under which the Factor H is eluted from the SiO₂ andthe serine protease or serine protease zymogen remains bound comprises aconductivity of less than about 10 mS/cm. In another embodiment of themethods described above, the solution condition under which the Factor His eluted from the SiO₂ and the serine protease or serine proteasezymogen remains bound comprises a conductivity between about 2 mS/cm andabout 20 mS/cm. In yet another embodiment of the methods describedabove, the solution condition under which the Factor H is eluted fromthe SiO₂ and the serine protease or serine protease zymogen remainsbound comprises a conductivity between about 2 mS/cm and about 10 mS/cm.

In a fourth aspect, the present invention provides a method forpreparing a Factor H composition having a reduced amount of a serineprotease or a serine protease zymogen, the method comprising the stepsof: (a) contacting a composition containing Factor H and at least oneserine protease or serine protease zymogen with finely divided silicondioxide (SiO₂) under conditions suitable to bind the Factor H but notthe at least one serine protease or serine protease zymogen; (b)separating the SiO₂ from the composition; and (c) eluting the Factor Hfrom the SiO₂.

In certain embodiment of the methods described above, the solutioncondition under which the Factor H binds to the SiO₂ and the serineprotease or serine protease zymogen does not comprises a pH greater thanabout 6.0. In another embodiment, the solution condition under which theFactor H binds to the SiO₂ and the serine protease or serine proteasezymogen does not comprises a pH greater than about 6.5. In anotherembodiment, the solution condition under which the Factor H binds to theSiO₂ and the serine protease or serine protease zymogen does notcomprises a pH greater than about 7.0. In yet another embodiment, thesolution condition under which the Factor H binds to the SiO₂ and theserine protease or serine protease zymogen does not comprises a pH of atleast about 7.5.

In certain embodiment of the methods described above, the solutioncondition comprises a pH of no more than 11.0. In another embodiment,the solution condition comprises a pH of no more than 10.0. In anotherembodiment, the solution condition comprises a pH of no more than 9.0.

In certain embodiment of the methods described above, the solutioncondition under which the Factor H binds to the SiO₂ and the serineprotease or serine protease zymogen does not comprises a conductivity ofgreater than about 10 mS/cm. In another embodiment of the methodsdescribed above, the solution condition under which the Factor H bindsto the SiO₂ and the serine protease or serine protease zymogen does notcomprises a conductivity of greater than about 20 mS/cm. In anotherembodiment of the methods described above, the solution condition underwhich the Factor H binds to the SiO₂ and the serine protease or serineprotease zymogen does not comprises a conductivity between about 10mS/cm and about 50 mS/cm. In yet another embodiment of the methodsdescribed above, the solution condition under which the Factor H bindsto the SiO₂ and the serine protease or serine protease zymogen does notcomprises a conductivity between about 20 mS/cm and about 50 mS/cm.

In a fifth aspect, the present invention provides a method for preparinga Factor H composition having a reduced amount of a serine protease or aserine protease zymogen, the method comprising the steps of: (a)contacting a composition containing Factor H and at least one serineprotease or serine protease zymogen with finely divided silicon dioxide(SiO₂) under conditions suitable to bind the at least one serineprotease or serine protease zymogen but not the Factor H and (b)separating the SiO₂ from the composition.

In certain embodiment of the methods described above, the solutioncondition under which the serine protease or serine protease zymogenbinds to the SiO₂ and the Factor H does not comprises a pH greater thanabout 6.0. In another embodiment, the solution condition under which theserine protease or serine protease zymogen binds to the SiO₂ and theFactor H does not comprises a pH greater than about 6.5. In anotherembodiment, the solution condition under which the serine protease orserine protease zymogen binds to the SiO₂ and the Factor H does notcomprises a pH greater than about 7.0. In yet another embodiment, thesolution condition under which the serine protease or serine proteasezymogen binds to the SiO₂ and the Factor H does not comprises a pH of atleast about 7.5.

In certain embodiment of the methods described above, the solutioncondition comprises a pH of no more than 11.0. In another embodiment,the solution condition comprises a pH of no more than 10.0. In anotherembodiment, the solution condition comprises a pH of no more than 9.0.

In certain embodiment of the methods described above, the solutioncondition under which the serine protease or serine protease zymogenbinds to the SiO₂ and the Factor H does not comprises a conductivity ofless than about 20 mS/cm. In another embodiment, the solution conditionunder which the serine protease or serine protease zymogen binds to theSiO₂ and the Factor H does not comprises a conductivity of less thanabout 10 mS/cm. In another embodiment of the methods described above,the solution condition under which the serine protease or serineprotease zymogen binds to the SiO₂ and the Factor H does not comprises aconductivity between about 2 mS/cm and about 20 mS/cm. In yet anotherembodiment of the methods described above, the solution condition underwhich the serine protease or serine protease zymogen binds to the SiO₂and the Factor H does not comprises a conductivity between about 2 mS/cmand about 10 mS/cm.

In a sixth aspect, the present invention provides a method for preparingan inter-alpha-trypsin inhibitor (IαI) composition having reduced serineprotease activity, the method comprising the steps of: (a) contacting asolution containing IαI and at least one serine protease with finelydivided silicon dioxide (SiO₂) under conditions suitable to bind the IαIand at least one serine protease; (b) separating the SiO₂ from thecomposition; (c) eluting the serine protease from the SiO₂ underconditions in which the IαI remains bound; and (d) eluting the IαI fromthe SiO₂.

In a seventh aspect, the present invention provides a method forpreparing an inter-alpha-trypsin inhibitor (IαI) composition havingreduced serine protease activity, the method comprising the steps of:(a) contacting a solution containing IαI and at least one serineprotease with finely divided silicon dioxide (SiO₂) under conditionssuitable to bind the IαI and at least one serine protease; (b)separating the SiO₂ from the composition; and (c) eluting the IαI fromthe SiO₂ under conditions in which the serine protease remains bound tothe SiO₂.

In an eighth aspect, the present invention provides a method forpreparing an inter-alpha-trypsin inhibitor (IαI) composition havingreduced serine protease activity, the method comprising the steps of:(a) contacting a solution containing IαI and at least one serineprotease with finely divided silicon dioxide (SiO₂) under conditionssuitable to bind the IαI but not the least one serine protease; (b)separating the SiO₂ from the composition; and (c) eluting the IαI fromthe SiO₂.

In a ninth aspect, the present invention provides a method for preparinga inter-alpha-trypsin inhibitor (IαI) composition having reduced serineprotease activity, the method comprising the steps of: (a) contacting asolution containing inter-alpha-trypsin inhibitor (IαI) and at least oneserine protease with finely divided silicon dioxide (SiO₂) underconditions suitable to bind the serine protease but not theinter-alpha-trypsin inhibitor (IαI); and (b) separating the SiO₂ fromthe composition.

In certain embodiments of the aspects described above, the compositionis contacted with SiO₂ at a final concentration of at least 1 g SiO₂/gprotein. In another embodiment of the aspects described above, thecomposition is contacted with SiO₂ at a final concentration of at least2 g SiO₂/g protein. In another embodiment of the aspects describedabove, the composition is contacted with SiO₂ at a final concentrationof at least 2.5 g SiO₂/g protein.

In certain embodiments of the aspects described above, the serineprotease or serine protease zymogen is Factor XI. In another embodimentof the aspects described above, the serine protease or serine proteasezymogen is Factor XIa. In another embodiment of the aspects describedabove, the serine protease or serine protease zymogen is Factor XII. Inyet another embodiment of the aspects described above, the serineprotease or serine protease zymogen is Factor XIIa.

In a tenth aspect, the present invention provides a plasma-derivedprotein composition prepared by a process comprising a method forreducing serine protease activity according to any one of the aspectsdescribed above. In one embodiment, the composition is formulated foradministration to a subject. In a specific embodiment, the compositionis formulated for intravenous, intramuscular, or subcutaneousadministration. In one embodiment, the composition is aqueous. Inanother embodiment, the composition is lyophilized.

In an eleventh aspect, the present invention provides a method fortreating a disease associated with aberrant activity of a plasma proteinin a subject in need thereof, the method comprising administering aplasma-derived protein composition according to the aspect outlinedabove. In certain embodiments, the composition comprises aplasma-derived protein is selected from an immunoglobulin (Ig), albumin,alpha-1-antitrypsin (A1PI), butyrylcholinesterase, a protein of thecomplement system, and an inter-alpha-trypsin inhibitor (IαI).

In a twelfth aspect, the present invention provides a method forpreparing a Factor H composition comprising the steps of (a) contactinga suspended plasma precipitate fraction containing Factor H with finelydivided silicon dioxide (SiO₂), (b) washing the SiO₂ with a wash buffercomprising a low pH and a low conductivity, and (c) eluting Factor Hfrom the SiO₂ with an elution buffer comprising a pH between 7.0 and 8.0and a conductivity of at least 10 mS/cm. In specific embodiments, theplasma precipitate fraction containing Factor H is a Cohn fractionII+III precipitate, a Cohn fraction I+II+III precipitate, aKistler/Nitschmann Precipitate A, or a Kistler/Nitschmann Precipitate B.In certain embodiments, the methods further comprises one or moreadditional steps selected from (d) precipitating and removing at leastone impurity from the Factor H elution, (e) precipitating and recoveringFactor H from the enriched composition, (f) further enriching Factor Hby anion exchange chromatography, (g) further enriching Factor H byheparin affinity chromatography, (h) a dedicated viral inactivationstep, and (i) concentrating the enriched Factor H composition byultrafiltration/diafiltration.

In one embodiment, the present invention provides a method for reducingthe amount of a serine protease or a serine protease zymogen in aplasma-derived target protein composition, the method comprising thesteps of: (a) contacting the composition with finely divided silicondioxide (SiO2) under conditions suitable to bind at least one serineprotease or serine protease zymogen; and (b) separating the SiO2 fromthe composition to remove the bound serine protease, wherein the atleast one serine protease or serine protease zymogen is Factor XIa(FXIa), Factor XIIa (FXIIa), Factor XI (FXI), or Factor XII (FXII).

In a specific embodiment of the methods described above, the methodfurther comprises the step of performing a first target proteinenrichment step to form a first enriched composition, prior tocontacting the composition with finely divided silicon dioxide (SiO2).In one embodiment, the first target protein enrichment step is a proteinprecipitation step. In a specific embodiment, the protein precipitationstep is an alcohol fractionation step. In another specific embodiment,the first target protein enrichment step is anultrafiltration/diafiltration step.

In a specific embodiment of the methods described above, the methodfurther comprises the step of performing a second target proteinenrichment step prior to contacting the enriched composition with finelydivided silicon dioxide (SiO2). In one embodiment, the second targetprotein enrichment step is a protein precipitation step. In a specificembodiment, the protein precipitation step is an alcohol fractionationstep. In one embodiment, the second target protein enrichment step is anultrafiltration/diafiltration step. In one embodiment, the second targetprotein enrichment step is a chromatographic enrichment step.

In a specific embodiment of the methods described above, the methodfurther comprises the step of performing a third target proteinenrichment step after contacting the composition with finely dividedsilicon dioxide (SiO2). In one embodiment, the third target proteinenrichment step is a protein precipitation step. In a specificembodiment, the protein precipitation step is an alcohol fractionationstep. In one embodiment, the third target protein enrichment step is anultrafiltration/diafiltration step. In one embodiment, the third targetprotein enrichment step is a chromatographic enrichment step.

In a specific embodiment of the methods described above, thechromatographic enrichment step comprises the sub-steps of: (i)contacting the plasma-derived target protein composition with achromatographic resin under conditions suitable to bind theplasma-derived target protein; and (ii) eluting the plasma-derivedtarget protein from the chromatographic resin. In one specificembodiment, at least one impurity does not bind to the chromatographicresin in sub-step (i). In another specific embodiment, at least oneimpurity binds to the chromatographic resin in sub-step (i), but is noteluted from the chromatographic resin in sub-step (ii).

In a specific embodiment of the methods described above, thechromatographic enrichment step comprises the sub-steps of: (i)contacting the first enriched plasma-derived target protein compositionwith a chromatographic resin under conditions suitable to bind at leastone impurity; and (ii) separating the resin from the plasma-derivedprotein composition, wherein the plasma-derived target protein does notbind to the chromatographic resin in sub-step (i).

In a specific embodiment of the methods described above, thechromatographic resin is selected from the group consisting of an anionexchange resin, a cation exchange resin, a hydrophobic interactionresin, a mixed mode resin, a hydroxyapatite resin, a ligand affinityresin, an immunoaffinity resin, and a size exclusion resin. In oneembodiment, the chromatographic resin is an anion exchange resin. In oneembodiment, the chromatographic resin is a cation exchange resin. In oneembodiment, the chromatographic resin is a hydrophobic interactionresin. In one embodiment, the chromatographic resin is a mixed moderesin. In one embodiment, the chromatographic resin is a hydroxyapatiteresin. In one embodiment, the chromatographic resin is a ligand affinityresin. In one embodiment, the chromatographic resin is an immunoaffinityresin. In one embodiment, the chromatographic enrichment step comprisesseparating at least one impurity from the target protein by size and/orshape using size exclusion chromatography.

In a specific embodiment of the methods described above, theplasma-derived target protein is selected from an immunoglobulin (Ig),albumin, alpha-1-antitrypsin (A1PI), butyrylcholinesterase, a protein ofthe complement system, and an inter-alpha-trypsin inhibitor (IαI). Inone embodiment, the plasma-derived protein is an immunoglobulin (Ig). Inone embodiment, the plasma-derived protein is albumin. In oneembodiment, the plasma-derived protein is alpha-1-antitrypsin. In oneembodiment, the plasma-derived protein is butyrylcholinesterase. In oneembodiment, the plasma-derived protein is a protein of the complementsystem. In one embodiment, the protein of the complement system isselected from the group consisting of Factor H (FH), Factor D,complement protein C3, and C4 binding protein. In one embodiment, theplasma-derived protein is an inter-alpha-trypsin inhibitor.

In a specific embodiment of the methods described above, theplasma-derived target protein composition is a manufacturingintermediate.

In one embodiment, the present invention provides a method for preparinga plasma-derived Factor H composition having a reduced amount of aserine protease or a serine protease zymogen, the method comprising thesteps of: (a) contacting a composition containing Factor H and at leastone serine protease or serine protease zymogen with finely dividedsilicon dioxide (SiO2) under conditions suitable to bind the Factor Hand at least one serine protease or serine protease zymogen; (b)separating the SiO2 from the composition; (c) eluting the serineprotease or serine protease zymogen from the SiO2 under a solutioncondition in which a substantial fraction of Factor H remains bound; and(d) eluting the Factor H from the SiO2.

In a specific embodiment of the methods described above, the solutioncondition under which the Factor H and at least one serine protease orserine protease zymogen bind the SiO2 comprises a pH below 7.0 and aconductivity of less than 11 mS/cm.

In a specific embodiment of the methods described above, the solutioncondition under which the Factor H and at least one serine protease orserine protease zymogen bind the SiO2 comprises a pH between 4.5 and 6.5and a conductivity of less than 6 mS/cm.

In a specific embodiment of the methods described above, the solutioncondition under which the Factor H and at least one serine protease orserine protease zymogen bind the SiO2 comprises a pH between 4.5 and 6.5and a conductivity between 0.5 mS/cm and 5 mS/cm.

In a specific embodiment of the methods described above, the solutioncondition under which the serine protease or serine protease zymogen iseluted from the SiO2 and a substantial fraction of the Factor H remainsbound comprises a pH below 7.0 and a conductivity of less than 11 mS/cm.

In a specific embodiment of the methods described above, the solutioncondition under which the serine protease or serine protease zymogen iseluted from the SiO2 and a substantial fraction of the Factor H remainsbound comprises a pH between 4.5 and 6.5 and a conductivity of less than6 mS/cm.

In a specific embodiment of the methods described above, the solutioncondition under which the serine protease or serine protease zymogen iseluted from the SiO2 and a substantial fraction of the Factor H remainsbound comprises a pH between 5.0 and 6.5 and a conductivity between 0.5mS/cm and 5 mS/cm.

In a specific embodiment of the methods described above, the solutioncondition under which the Factor H is eluted from the SiO2 comprises anionic strength of at least 6 mS/cm.

In a specific embodiment of the methods described above, the solutioncondition under which the Factor H is eluted from the SiO2 comprises anionic strength of at least 11 mS/cm.

In a specific embodiment of the methods described above, the solutioncondition under which the Factor H is eluted from the SiO2 comprises apH between 5.0 and 7.0.

In a specific embodiment of the methods described above, the solutioncondition under which the Factor H is eluted from the SiO2 comprises apH between 7.0 and 8.0 and an ionic strength of between 4 mS/cm and 7mS/cm.

In one embodiment, the present invention provides a method for preparinga Factor H composition having a reduced amount of a serine protease or aserine protease zymogen, the method comprising the steps of: (a)contacting a composition containing Factor H and at least one serineprotease or serine protease zymogen with finely divided silicon dioxide(SiO2) under conditions suitable to bind the Factor H and at least oneserine protease; (b) separating the SiO2 from the composition; and (c)eluting the Factor H from the SiO2 under conditions in which asubstantial fraction of the serine protease or serine protease zymogenremains bound to the SiO2.

In a specific embodiment of the methods described above, the solutioncondition under which the Factor H and at least one serine protease orserine protease zymogen bind the SiO2 comprises a pH below 7.0 and aconductivity of less than 11 mS/cm.

In a specific embodiment of the methods described above, the solutioncondition under which the serine protease or serine protease zymogen iseluted from the SiO2 and a substantial fraction of the Factor H remainsbound comprises a pH between 4.5 and 6.5 and a conductivity of less than6 mS/cm.

In a specific embodiment of the methods described above, the solutioncondition suitable to bind the Factor H and at least one serine proteaseor serine protease zymogen comprises a pH between 5.0 and 6.0 and aconductivity of between 0.5 mS/cm and 5.0 mS/cm.

In a specific embodiment of the methods described above, the solutioncondition under which the Factor H is eluted from the SiO2 and asubstantial fraction of the serine protease or serine protease zymogenremains bound comprises a pH between 5.0 and 7.0 and a conductivity ofat least 11 mS/cm.

In a specific embodiment of the methods described above, the solutioncondition under which the Factor H is eluted from the SiO2 and asubstantial fraction of the serine protease or serine protease zymogenremains bound comprises a pH between 7.0 and 8.0 and a conductivity ofbetween 2 mS/cm and 10 mS/cm.

In a specific embodiment of the methods described above, theconductivity of the solution condition is between 4 mS/cm and 7 mS/cm.

In a specific embodiment of the methods described above, theconductivity of the solution condition is between 5 mS/cm and 6 mS/cm.

In one embodiment, the present invention provides method for preparing aFactor H composition having a reduced amount of a serine protease or aserine protease zymogen, the method comprising the steps of: (a)contacting a composition containing Factor H and at least one serineprotease or serine protease zymogen with finely divided silicon dioxide(SiO2) under conditions suitable to bind the Factor H but not asubstantial fraction of the at least one serine protease or serineprotease zymogen; (b) separating the SiO2 from the composition; and

(c) eluting the Factor H from the SiO2.

In a specific embodiment of the methods described above, the solutioncondition under which the Factor H binds to the SiO2 and a substantialfraction of the serine protease or serine protease zymogen does not bindto the SiO2 comprises a pH of between 5.0 and 7.0 and a conductivity ofno more than 14 mS/cm.

In a specific embodiment of the methods described above, theconductivity of the solution condition is between 9 mS/cm and 14 mS/cm.

In one embodiment, the present invention provides a method for preparinga Factor H composition having a reduced amount of a serine protease or aserine protease zymogen, the method comprising the steps of: (a)contacting a composition containing Factor H and at least one serineprotease or serine protease zymogen with finely divided silicon dioxide(SiO2) under conditions suitable to bind the at least one serineprotease or serine protease zymogen but not a substantial fraction ofthe Factor H; and (b) separating the SiO2 from the composition.

In a specific embodiment of the methods described above, the solutioncondition under which the serine protease or serine protease zymogenbinds to the SiO2 and a substantial fraction of the Factor H does notbind to the SiO2 comprises a pH between 5.0 and 7.0 and a conductivityof at least 11 mS/cm.

In a specific embodiment of the methods described above, the solutioncondition under which the serine protease or serine protease zymogenbinds to the SiO2 and a substantial fraction of the Factor H does notbind to the SiO2 comprises a pH between 7.0 and 8.0 and a conductivityof between 2 mS/cm and 10 mS/cm.

In a specific embodiment of the methods described above, theconductivity of the solution condition is between 4 mS/cm and 7 mS/cm.

In a specific embodiment of the methods described above, theconductivity of the solution condition is between 5 mS/cm and 6 mS/cm.

In one embodiment, the present invention provides a method for preparinga Factor H composition, the method comprising the steps of: (a)contacting a composition containing Factor H and at least one serineprotease or serine protease zymogen with finely divided silicon dioxide(SiO2) under conditions suitable to bind the Factor H; (b) washing theSiO2 with a solution comprising a pH between 5.0 and 7.0 and aconductivity of less than 4 mS/cm; and (c) eluting Factor H from theSiO2 with a solution comprising a pH between 7.0 and 8.0 and aconductivity greater than 10 mS/cm.

In a specific embodiment of the methods described above, the solutionused to wash the SiO2 comprises a pH between 5.5 and 6.5.

In a specific embodiment of the methods described above, the solutionused to wash the SiO2 comprises a pH between of 6.0±0.2.

In a specific embodiment of the methods described above, the solutionused to elute Factor H comprises a conductivity of at least 20 mS/cm.

In a specific embodiment of the methods described above, the solutionused to elute Factor H comprises a conductivity of between 25 mS/cm and40 mS/cm.

In a specific embodiment of the methods described above, the startingcomposition containing Factor H is a suspended Cohn fraction II+IIIprecipitate, or equivalent fraction thereof.

In a specific embodiment of the methods described above, the startingcomposition containing Factor H is a suspended Kistler/NitschmannPrecipitate A, or equivalent fraction thereof.

In a specific embodiment of the methods described above, the startingcomposition containing Factor H is a suspended Kistler/NitschmannPrecipitate B, or equivalent fraction thereof.

In a specific embodiment of the methods described above, the methodfurther comprises the step of precipitating at least one impurity fromthe recovered Factor H solution, wherein Factor H is not precipitated.

In a specific embodiment of the methods described above, theprecipitation step is PEG precipitation.

In a specific embodiment of the methods described above, the PEGprecipitation comprises precipitation with PEG 4000 at a finalconcentration between 3% and 7%.

In a specific embodiment of the methods described above, the finalconcentration of PEG 4000 is 5±0.5%.

In a specific embodiment of the methods described above, the methodfurther comprises the step of precipitating Factor H from the recoveredFactor H solution.

In a specific embodiment of the methods described above, theprecipitation step is PEG precipitation.

In a specific embodiment of the methods described above, the PEGprecipitation comprises precipitation with PEG 4000 at a finalconcentration between 10% and 15%.

In a specific embodiment of the methods described above, the finalconcentration of PEG 4000 is 12±0.5%.

In a specific embodiment of the methods described above, the methodfurther comprises a step of enriching Factor H by chromatography.

In a specific embodiment of the methods described above, thechromatographic enrichment step comprises anion exchange chromatography.

In a specific embodiment of the methods described above, thechromatographic enrichment step comprises heparin affinitychromatography.

In a specific embodiment of the methods described above, thechromatographic enrichment step comprises anion exchange chromatographyfollowed by heparin affinity chromatography.

In a specific embodiment of the methods described above, the methodfurther comprises at least one dedicated viral removal or inactivationstep.

In a specific embodiment of the methods described above, the methodcomprises a nanofiltration step.

In a specific embodiment of the methods described above, the methodfurther comprises a step of concentrating the Factor H compositioncomprising ultrafiltration/diafiltration.

In one embodiment, the present invention provides a method for preparingan inter-alpha-trypsin inhibitor (IαI) composition having a reducedamount of a serine protease or a serine protease zymogen, the methodcomprising the steps of: (a) contacting a solution containing IαI and atleast one serine protease with finely divided silicon dioxide (SiO2)under conditions suitable to bind the IαI and at least one serineprotease; (b) separating the SiO2 from the composition; (c) eluting theserine protease or serine protease zymogen from the SiO2 underconditions in which a substantial fraction of the IαI remains bound; and(d) eluting the IαI from the SiO2.

In one embodiment, the present invention provides a method for preparingan inter-alpha-trypsin inhibitor (IαI) composition having a reducedamount of a serine protease or a serine protease zymogen, the methodcomprising the steps of: (a) contacting a solution containing IαI and atleast one serine protease with finely divided silicon dioxide (SiO2)under conditions suitable to bind the IαI and at least one serineprotease; (b) separating the SiO2 from the composition; and (c) elutingthe IαI from the SiO2 under conditions in which a substantial fractionof the serine protease or serine protease zymogen remains bound to theSiO2.

In one embodiment, the present invention provides a method for preparingan inter-alpha-trypsin inhibitor (IαI) composition having a reducedamount of a serine protease or a serine protease zymogen, the methodcomprising the steps of: (a) contacting a solution containing IαI and atleast one serine protease with finely divided silicon dioxide (SiO2)under conditions suitable to bind the IαI but not a substantial fractionof the least one serine protease or serine protease zymogen; (b)separating the SiO2 from the composition; and (c) eluting the IαI fromthe SiO2.

In one embodiment, the present invention provides a method for preparinga inter-alpha-trypsin inhibitor (IαI) composition having a reducedamount of a serine protease or a serine protease zymogen, the methodcomprising the steps of: (a) contacting a solution containinginter-alpha-trypsin inhibitor (IαI) and at least one serine proteasewith finely divided silicon dioxide (SiO2) under conditions suitable tobind the serine protease or serine protease zymogen but not theinter-alpha-trypsin inhibitor (IαI); and (b) separating the SiO2 fromthe composition.

In one embodiment, the present invention provides a method for preparingan Immunoglobulin G (IgG) composition having a reduced amount of aserine protease or a serine protease zymogen, the method comprising thesteps of: (a) precipitating a cryo-poor plasma fraction, in a firstprecipitation step, with between about 6% and about 10% alcohol at a pHof between about 7.0 and about 7.5 to obtain a first precipitate and afirst supernatant; (b) precipitating IgG from the first supernatant, ina second precipitation step, with between about 20% and about 25%alcohol at a pH of between about 6.7 and about 7.3 to form a secondprecipitate; (c) re-suspending the second precipitate to form asuspension; (d) contacting the suspension with finely divided silicondioxide (SiO2) under a solution condition suitable to bind a serineprotease or serine protease zymogen; and (e) separating the SiO2 fromthe suspension to form a clarified suspension.

In a specific embodiment of the methods described above, the methodfurther comprises the steps of: (f) precipitating IgG from the clarifiedsuspension formed in step (e), in a third precipitation step, withbetween about 22% and about 28% alcohol at a pH of between about 6.7 andabout 7.3 to form a third precipitate; (g) re-suspending the thirdprecipitate to form a suspension; and (h) separating the solublefraction from the suspension formed in step (e), thereby forming anenriched IgG composition.

In a specific embodiment of the methods described above, the methodfurther comprises an anion exchange chromatography enrichment step.

In a specific embodiment of the methods described above, the methodfurther comprises a cation exchange chromatography enrichment step.

In a specific embodiment of the methods described above, the methodfurther comprises at least one dedicated viral inactivation or removalstep.

In a specific embodiment of the methods described above, the methodcomprises a solvent/detergent (S/D) viral inactivation step.

In a specific embodiment of the methods described above, the methodcomprises a nanofiltration step.

In a specific embodiment of the methods described above, the methodcomprises an incubation step at low pH.

In a specific embodiment of the methods described above, step (b)comprises adjusting the ethanol concentration of the first supernatantformed in step (a) to about 25% (v/v) at a temperature between about −7°C. and about −9° C.

In a specific embodiment of the methods described above, the temperatureis about −9° C.

In a specific embodiment of the methods described above, step (c)comprises re-suspending the precipitate of step (b) with a buffercontaining phosphate and acetate, wherein the pH of the buffer isadjusted with between 300 mL and 700 mL of glacial acetic acid per 1000L of buffer.

In a specific embodiment of the methods described above, step (d)comprises the addition SiO2 to a final concentration of between about0.02 grams per gram precipitate formed in step (b) and about 0.06 gramsper gram precipitate formed in step (b).

In a specific embodiment of the methods described above, the solutioncondition suitable to bind a serine protease or serine protease zymogencomprises a pH between 4.5 and 6.0 and a conductivity of between 0.1mS/cm and 3 mS/cm.

In a specific embodiment of the methods described above, the pH isbetween 4.9 and 5.3.

In a specific embodiment of the methods described above, theconductivity is between 0.5 mS/cm and 2 mS/cm.

In a specific embodiment of the methods described above, step (e)comprises the sub-steps of: (i) washing the filter press with at least 3filter press dead volumes of a buffer containing phosphate and acetate,wherein the pH of the buffer is adjusted with between 50 mL and 200 mLof glacial acetic acid per 1000 L of buffer, thereby forming a washsolution; and (ii) combining the filtrate of step (f) with the washsolution of step (g), thereby forming a solution.

In a specific embodiment of the methods described above, furthercomprising the sub-step of: (iii) treating the solution with adetergent.

In a specific embodiment of the methods described above, step (h)further comprises solvent and detergent (S/D) treatment of the enrichedIgG composition.

In a specific embodiment of the methods described above, the enrichedIgG composition obtained in step (h) contains at least 85% of the IgGcontent found in the cryo-poor plasma fraction used in step (a).

In a specific embodiment of the methods described above, the enrichedIgG composition obtained in step (h) contains at least 90% of the IgGcontent found in the cryo-poor plasma fraction used in step (a).

In a specific embodiment of the methods described above, the amount of aserine protease or a serine protease zymogen has been reduced by atleast 90%.

In a specific embodiment of the methods described above, the amount of aserine protease or a serine protease zymogen has been reduced by atleast 95%.

In a specific embodiment of the methods described above, the amount of aserine protease or a serine protease zymogen has been reduced by atleast 98%.

In a specific embodiment of the methods described above, the amount of aserine protease or a serine protease zymogen has been reduced by atleast 99%.

In a specific embodiment of the methods described above, the serineprotease or a serine protease zymogen is FXIa.

In a specific embodiment of the methods described above, the compositionis contacted with SiO2 at a final concentration of at least 1 g SiO2/gprotein.

In a specific embodiment of the methods described above, the compositionis contacted with SiO2 at a final concentration of at least 2 g SiO2/gprotein.

In a specific embodiment of the methods described above, the compositionis contacted with SiO2 at a final concentration of at least 2.5 g SiO2/gprotein.

In a specific embodiment of the methods described above, the serineprotease or serine protease zymogen is Factor XI.

In a specific embodiment of the methods described above, the serineprotease or serine protease zymogen is Factor XII.

In a specific embodiment of the methods described above, the serineprotease or serine protease zymogen is Factor XIa.

In a specific embodiment of the methods described above, the serineprotease or serine protease zymogen is Factor XIIa.

In one embodiment, the present invention provides a plasma-derivedprotein composition prepared by a process comprising a method forreducing serine protease activity according to any one of the precedingclaims.

In a specific embodiment of the compositions described above, thecomposition is formulated for administration to a subject.

In a specific embodiment of the compositions described above, thecomposition is formulated for intravenous, intramuscular, orsubcutaneous administration.

In a specific embodiment of the compositions described above, thecomposition is aqueous.

In a specific embodiment of the compositions described above, thecomposition is lyophilized.

In one embodiment, the present invention provides a method for treatinga disease associated with aberrant activity of a plasma protein in asubject in need thereof, the method comprising administering aplasma-derived protein composition according to any one of claims 124 to128. In one embodiment, the composition comprises a plasma-derivedprotein is selected from an immunoglobulin (Ig), albumin,alpha-1-antitrypsin (A1PI), butyrylcholinesterase, a protein of thecomplement system, and an inter-alpha-trypsin inhibitor (IαI).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Overview of an exemplary plasma fractionation scheme.

FIG. 2. Factor H content in select fractions of an industrial-scaleplasma protein fractionation, as measured by ELISA

FIG. 3. Illustration of Factor H and amidolytic activity (as measuredusing substrate CS2166) eluted from SiO₂ under solution conditions withvarying conductivities at pH 7.5.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Given the broad use of therapeutic plasma-derived blood proteincompositions, such as immune globulin compositions, blood coagulationfactors, coagulation factor inhibitors, and proteins of the complementsystem, ensuring the safety of these compositions is of paramountimportance. Recent concerns over the amidolytic content of thesecompositions paired with the occurrence of thromboembolic events inpatients being administered plasma-derived protein compositions, hashighlighted a need in the art for method for reducing serine proteases(e.g., FXIa and FXIIa) and serine protease zymogens (e.g., FXI and FXII)during the manufacturing of these biologics. Advantageously, the presentinvention is based at least in part on the surprising finding thatfinely divided silicon dioxide (SiO₂) can be used to bind serineproteases and serine protease zymogens present in plasma-derived proteincompositions. As such, methods are provided herein for reducing theconcentration of serine proteases and serine protease zymogens duringthe manufacture of plasma-derived protein compositions.

In certain aspects, the present invention provides manufacturing methodsbased on the surprising finding that finely divided silicon dioxide(SiO₂) can be used to remove significant amounts of serine protease(e.g., FXIa and FXIIa) and serine protease zymogen (e.g., FXI and FXII)from plasma-derived protein solutions. As such, the methods providedherein may be easily integrated into existing manufacturing procedures,for example, the fractionation of pooled plasma samples, preferablyhuman plasma samples, by ethanol in the cold (reviewed in Schultze H E,Heremans J F; Molecular Biology of Human Proteins. Volume I: Nature andMetabolism of Extracellular Proteins 1966, Elsevier Publishing Company;p. 236-317). However, the methods provided herein are in no way limitedin their use to manufacturing methods including ethanol fractionation.Other methodologies for the purification of plasma-derived proteins arealso compatible with the methods provided herein, for example, polymer(e.g., PEG) fractionation and chromatographic methodologies (e.g., anionand/or cation exchange chromatography, affinity chromatography,immuno-affinity chromatography, size exclusion chromatography,hydrophobic interaction chromatography, mixed mode chromatography, andthe like).

Furthermore, unlike other biologics that are produced via recombinantexpression of DNA vectors in host cell lines, plasma-derived proteinsare fractionated from human blood and plasma donations. Thus, the supplyof these products cannot be increased by simply increasing the volume ofproduction. Rather the level of commercially available blood products islimited by the available supply of blood and plasma donations. Thisdynamic results in a shortage in the availability of raw human plasmafor the manufacture of new plasma-derived blood factors that have lesserestablished commercial markets, including Complement Factor H (CFH) andinter-alpha-trypsin inhibitor proteins (IαIp).

Due to the lack of plasma available for the manufacture of newplasma-derived products, their manufacture must be integrated into theexisting framework of the established manufacturing processes forplasma-derived products such as immunoglobulins and albumin. Factor H,implicated as a potential therapeutic for AMD, aHUS, and MPGN, amongother conditions, is one such plasma-derived blood product that isgaining the attention of physicians. However, due to the resourcesdevoted to, for example, IgG gamma globulin manufacture, methods areneeded for the manufacture of Factor H that can be introduced into theexisting manufacturing schemes. Several methods have been suggested toachieve just this, however, many of these proposed solutions requiremodification of the existing manufacturing scheme for establishedproducts. Such changes will require new regulatory approvals for theestablished products and may even result in alterations of thecharacteristics of the established products.

For example, WO 2007/066017 describes methods for the production ofFactor H preparations from the supernatant of a cryoprecipitate. Thedisclosed method consists of preparing a supernatant of acryoprecipitate, submitting the supernatant to anion exchangechromatography (AEC), submitting the flow through from the AEC toheparin affinity chromatography (HAC), submitting the relevant eluatefrom the HAC to strong cation exchange chromatography (CEC), submittingthe relevant eluate from the CEC to strong anion exchange chromatography(sAEC) and eluting the Factor H from the sAEC. Disadvantageously,cryoprecipitate supernatants are common intermediate fractions in themanufacturing processes of many commercially important plasma-derivedblood products, including IgG gamma globulins (IVIG and subcutaneous)and albumin. Submitting this fraction to chromatography steps will alterthe cryoprecipitate supernatant and would require that the manufacturingprocesses of the established downstream blood products be adapted inunknown fashions. In addition to requiring a complete revalidation andpossible redesign of these manufacturing processes, regulatoryre-approval of the manufacturing procedures from key regulatory agenciesis needed.

Likewise, WO 2008/113589 describes methods for the production of FactorH preparations from human plasma. Specifically, this publicationdescribes the purification of Factor H from three known plasmaprocessing fractions, namely a Cohn-Oncley Fraction I supernatant, aCohn-Oncley Fraction III precipitate, and a Kistler/NitschmannPrecipitate B fraction. With respect to the first method, WO 2008/113589discloses that Factor H can be removed from a Cohn-Oncley Fraction Isupernatant by the addition of a heparin affinity chromatography step.Disadvantageously, the Cohn-Oncley Fraction I supernatant is a commonintermediate fraction in the manufacturing processes of manycommercially important plasma-derived blood products, including IgGgamma globulins (IVIG and subcutaneous) and albumin. Similarly, manyimmunoglobulin (e.g., IgG, IVIG, etc.) manufacturing processes do notrely on Cohn-Oncley Fraction III precipitation or Kistler/NitschmannPrecipitate B steps, for example Gammagard® Liquid and Kiovig (BaxterInternational Inc.). The disadvantage of the introduction of additionalsteps, such as a heparin affinity chromatography, Fraction IIIprecipitation, or Precipitate B steps, into the manufacturing schemes ofestablished blood products, as outlined above, is that it requiresrevalidation of the manufacturing procedure, regulatory re-approval ofthe manufacturing procedures from key regulatory agencies, and mayfurther have unforeseen consequences for the yield and/or purity of theotherwise established product.

As such, a need remains in the art for methods of manufacturing Factor Hthat do not require the use of additional input plasma or the redesignand regulatory re-approval of existing manufacturing processes forcommercially important plasma-derived blood products, such as albuminand IgG gamma globulins for intravenous (IVIG) or subcutaneousadministration. Advantageously, the present invention is based at leastin part on the surprising discovery that Factor H, serine proteases, andserine protease zymogens can be simultaneously bound to finely dividedsilicon dioxide (SiO₂) thereby separating serine proteases and serineprotease zymogen from a first protein of interest not bound to (e.g.,IgG) and then separated by differentially eluting Factor H and theserine protease and serine protease zymogens from the SiO₂. Similarly,the present invention is based at least in part on the surprisingdiscovery that IαIp, serine proteases, and serine protease zymogens canbe simultaneously bound to finely divided silicon dioxide (SiO₂) andthen separated by differentially eluting IαIp and the serine proteaseand serine protease zymogens from the SiO₂.

II. Definitions

As used herein, “Factor H” refers to a protein component of thealternative pathway of complement encoded by the complement factor Hgene (for example, CFH; NM000186; GeneID:3075; UniProt ID P08603;Ripoche et al., Biochem. J. 249:593-602 (1988)). Factor H is translatedas a 1,213 amino acid precursor polypeptide which is processed byremoval of an 18 amino acid signal peptide, resulting in the matureFactor H protein (amino acids 19-1231). As used in the presentinvention, Factor H encompasses any natural variants, alternativesequences, isoforms or mutant proteins that can be found in a plasmasample, for example a human plasma sample. Examples of Factor Hmutations found in the human population include, without limitation,Y402H; V62I; R78G; R127L; 4224; Q400K; C431S; T493R; C536R; 1551T;R567G; C630W; C673S; C673Y; E850K; S890I; H893R; C915S; E936D; Q950H;Y951H; T956M; C959Y; W978C; N997T; V1007I; V1007L; A1010T; T1017I;Y1021F; C1043R; N1050Y; 11059T; Q1076R; R1078S; D1119G; V1134G; Y1142D;Q1143E; W1157R; C1163W; W1183L; W1183R; T1184R; L1189R; S1191L; G1194D;V1197A; E1198A; F1199S; R1210C; R1215G; R1215Q; YPTCAKR1225:1231FQS; andP1226S. Many of the these mutations have been found to be associatedwith a variety of diseases and disorders, including, atypical haemolyticuremic syndrome (aHUS), age-related macular degeneration (AMD),membranoproliferative glomulonephritis type II (MPGNII), CFH deficiency,and basal laminar drusen. Factor H also includes proteins containingpost-translational modifications. For example, Factor H is believed tobe modified by N-acetylglucosamine (GlcNAc) at residues 529, 718, 802,822, 882, 911, 1029, and 1095.

As used herein, “Inter-alpha-Inhibitor proteins” or “IaIp” refers to afamily of plasma protease inhibitors comprised of polypeptides encodedby one or more of the Alpha-1-microglobulin/bikunin precursor gene(AMBP; UniGene ID:231948, bikunin polypeptide), Inter-alpha (globulin)inhibitor H1 gene (ITIH1; UniGene ID:224173, H1 polypeptide),Inter-alpha (globulin) inhibitor H2 gene (ITIH2; Unigene ID:139782, H2polypeptide), Inter-alpha (globulin) inhibitor H3 gene (ITIH3; UniGeneID:140017, H3 polypeptide), or Inter-alpha (globulin) inhibitor H4(plasma Kallikrein-sensitive glycoprotein, H4 polypeptide) gene (ITIH4;UniGene ID:3321613). Exemplary IaIp protease inhibitors include, withoutlimitation, IaI (bikunin, H1, and H2 polypeptides); PaI (bikunin and H3polypeptides), IaLI (bikunin and H2 polypeptides), IaIH4P (H4polypeptide), and bikunin (Salier, J, et al., supra).

As used herein, “cryo-poor plasma” refers to the supernatant createdafter the removal of cryo-precipitate formed by thawing plasma or pooledplasma at temperatures near freezing, e.g., at temperatures below about10° C., preferably at a temperature no higher than about 6° C. In thecontext of the present invention, plasma may refer interchangeably torecovered plasma (i.e., plasma that has been separated from whole bloodex vivo) or source plasma (i.e., plasma collected via plasmapheresis).Cryo-precipitation is commonly performed, for example, by thawingpreviously frozen pooled plasma, which has already been assayed forsafety and quality considerations, although fresh plasma may also beused. After complete thawing of the frozen plasma at low temperature,separation of the solid cryo-precipitates from the liquid supernatant isperformed in the cold (e.g., ≦6° C.) by centrifugation of filtration.

As used herein, a “Cohn pool” refers to the starting material used forthe fractionation of a plasma sample or pool of plasma samples. Cohnpools include whole plasma, cryo-poor plasma samples, and pools ofcryo-poor plasma samples that may or may not have been subjected to apre-processing step. In certain embodiments, a Cohn pool is a cryo-poorplasma sample from which one or more blood factor have been removed in apre-processing step, for example, adsorption onto a solid phase (e.g.,aluminum hydroxide, finely divided silicon dioxide, etc.), orchromatographic step (e.g., ion exchange or heparin affinitychromatography). Various blood factors, including but not limited toFactor Eight Inhibitor Bypass Activity (FEIBA), Factor IX-complex,Factor VII-concentrate, or Antithrombin III-complex, may be isolatedfrom the cryo-poor plasma sample to form a Cohn pool.

As used herein, a “Fraction II+III filter cake” refers to a solid phaserecovered after the filtration or centrifugation of a Cohn-Oncley orequivalent Fraction II+III paste suspension. In a preferred embodiment,a Fraction II+III suspension will be treated with an adsorptivematerial, for example, finely divided silicon dioxide, to removeimpurities such as lipids, fibrinogen, amidolytic activity, prekallikrenactivity, and lipoproteins. In another preferred embodiment, filter aidmay be added to the Fraction II+III suspension prior to centrifugationor filtration. In a most preferred embodiment, a Fraction II+IIIsuspension will be treated with both an adsorptive material and a filteraid prior to centrifugation or filtration. Upon separation of theclarified Fraction II+III suspension supernatant, the recovered solidphase material is referred to as the Fraction II+III filter cake.

As used herein, “finely divided silicon dioxide” or “finely dividedsilica” refers to an oxide of silicon having the formula SiO₂,manufactured in a fashion that allows for the adsorption of Factor Honto its surface. Exemplary forms of finely divided silicon dioxidesuitable for use in the methods of the present invention include,without limitation, fumed silica, pyrogenic silica, Aerosil®,Cab-O-Sil™, colloidal silica, diatomaceous earth, and the like. In apreferred embodiment, a commercial hydrophilic fumed silica product isused for the methods provided herein. Non-limiting examples of theseproducts include those marketed by Evonik Industries under the tradename Aerosil® (e.g., Aerosil 90, Aerosil 130, Aerosil 150, Aerosil 200,Aerosil 300, Aerosil 380, Aerosil OX 50, Aerosil EG 50, Aerosil TT 600,Aerosil 200 SP, Aerosil 300 SP, and Aerosil 300/30).

As used herein, a “disease or disorder associated with Factor Hdysfunction” refers to any disease, disorder, or condition in a subjectthat is caused by, characterized by, or results in a reduced level ofFactor H activity in the subject. For purposes of the present invention,Factor H activity may refer to the ability of Factor H to bind a proteinor ligand, for example, C3b, C3bBb, C3b2Bb, csbC3b, complement factor B(CFB), C-reactive protein, endothelial cells, glycosaminoglycans (GAGs),or alternatively, may refer to its Factor I cofactor activity or itsability to accelerate the irreversible dissociation of C3bBb and C3b2Bb.In one embodiment, a disease or disorder associated with Factor Hdysfunction results in a C3 deficiency and susceptibility to bacterialinfections. In some instances, diseases or disorders associated withFactor H dysfunction include conditions that are caused by or linked tomutations and polymorphism in the CFH gene encoding Factor H (forreview, see, Barlow et al., Adv Exp Med Biol. 2008; 632:117-42, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes). Diseases that have been linked to mutations orpolymorphisms in the CFH gene include, without limitation, Factor Hdeficiency, atypical haemolytic uremic syndrome (aHUS), age-relatedmacular degeneration (AMD), membranoproliferative glomulonephritis typeII (MPGNII; de Cordoba and de Jorge, Clinical and ExperimentalImmunology 151, 1-13 (2008)), myocardial infarction (Kardys et al.,Journal of the American College of Cardiology 47, 1568-1575 (2006);Mooijaart et al., Experimental Gerontology 42, 1116-1122 (2007); Nicaudet al., Journal of Molecular Medicine 85, 771-775 (2007); Pai et al.,European Heart Journal 28, 1297-1303 (2007); Stark et al., ClinicalScience (Lond) 113, 213-218 (2007)), coronary heart disease/coronaryartery disease (CAD/CHD; (Meng et al., BMC Medical Genetics 8, 62(2007); Pulido et al., Mayo Clinic Proceedings 82, 301-307 (2007); Topolet al., Human Molecular Genetics 15 Spec No 2, R117-R123 (2006)), andAlzheimer's disease (Hamilton et al., Neuromolecular Medicine 9, 331-334(2007); Zetterberg et al., American Journal of Ophthalmology 143,1059-1060 (2007)). The disclosures of the forgoing references describingthe associations between mutations and polymorphisms in the CFH gene anddiseases associated with Factor H dysfunction are herein incorporated byreference in their entireties for all purposes.

As used herein, a “disease or disorder associated with abnormalalternative pathway complement activity” refers to a disease, disorder,or condition that results from uncontrolled or aberrant activation ofthe alternative pathway of complement. Generally, uncontrolled oraberrant activation of the alternative pathway of complement can resultin bystander damage of host cells and tissues, as well as a depletion ofC3 and corresponding susceptibility to pathogenic infections (e.g.,fungal, bacterial, viral, and protistal). Examples of diseases anddisorders associated with abnormal alternative pathway complementactivity include, without limitation, various autoimmune diseases (suchas rheumatoid arthritis, IgA nephropathy, asthma, systemic lupuserythematosus, multiple sclerosis, Anti-Phospholipid syndrome,ANCA-associated vasculitis, pemphigus, uveitis, myathemia gravis,Hashimoto's thyroiditis), Renal diseases (such as IgA nephropathy,hemolytic uremic syndrome, membranoproliferative glomerulonephritis)other disease such as asthma, Alzheimer disease, adult maculardegeneration, proximal nocturnal hemoglobinuria, abdominal aorticaneurism, ischemia, and sepsis.

As used herein, the term “ultrafiltration (UF)” encompasses a variety ofmembrane filtration methods in which hydrostatic pressure forces aliquid against a semi-permeable membrane. Suspended solids and solutesof high molecular weight are retained, while water and low molecularweight solutes pass through the membrane. This separation process isoften used for purifying and concentrating macromolecular (10³-10⁶ Da)solutions, especially protein solutions. A number of ultrafiltrationmembranes are available depending on the size of the molecules theyretain. Ultrafiltration is typically characterized by a membrane poresize between 1 and 1000 kDa and operating pressures between 0.01 and 10bar.

As used herein, the term “diafiltration” is performed with the same or asimilar membrane as ultrafiltration and is typically performed in atangential flow filtration mode. During diafiltration, buffer isintroduced into the recycle tank while filtrate is removed from the unitoperation. In processes where the product is in the retentate (forexample, Factor H), diafiltration is particularly useful for separatingprotein from small molecules like sugars and salts. In certain cases,diafiltration can be used to exchange the solution, buffer, orindividual components of a buffering system.

As used herein, the term “mixing” describes an act of causing equaldistribution of two or more distinct compounds or substances in asolution or suspension by any form of agitation. Complete equaldistribution of all ingredients in a solution or suspension is notrequired as a result of “mixing” as the term is used in thisapplication.

As used herein, the term “solvent” encompasses any liquid substancecapable of dissolving or dispersing one or more other substances. Asolvent may be inorganic in nature, such as water, or it may be anorganic liquid, such as ethanol, acetone, methyl acetate, ethyl acetate,hexane, petrol ether, etc. As used in the term “solvent detergenttreatment,” solvent denotes an organic solvent (e.g., tri-N-butylphosphate), which is part of the solvent detergent mixture used toinactivate lipid-enveloped viruses in solution.

As used herein, the term “detergent” is used in this applicationinterchangeably with the term “surfactant” or “surface acting agent.”Surfactants are typically organic compounds that are amphiphilic, i.e.,containing both hydrophobic groups (“tails”) and hydrophilic groups(“heads”), which render surfactants soluble in both organic solvents andwater. A surfactant can be classified by the presence of formallycharged groups in its head. A non-ionic surfactant has no charge groupsin its head, whereas an ionic surfactant carries a net charge in itshead. A zwitterionic surfactant contains a head with two oppositelycharged groups. Some examples of common surfactants include: Anionic(based on sulfate, sulfonate or carboxylate anions): perfluorooctanoate(PFOA or PFO), perfluorooctanesulfonate (PFOS), sodium dodecyl sulfate(SDS), ammonium lauryl sulfate, and other alkyl sulfate salts, sodiumlaureth sulfate (also known as sodium lauryl ether sulfate, or SLES),alkyl benzene sulfonate; cationic (based on quaternary ammoniumcations): cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyltrimethyl ammonium bromide, and other alkyltrimethylammonium salts,cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA),benzalkonium chloride (BAC), benzethonium chloride (BZT); Long chainfatty acids and their salts: including caprylate, caprylic acid,heptanoat, hexanoic acid, heptanoic acid, nanoic acid, decanoic acid,and the like; Zwitterionic (amphoteric): dodecyl betaine; cocamidopropylbetaine; coco ampho glycinate; nonionic: alkyl poly(ethylene oxide),alkylphenol poly(ethylene oxide), copolymers of poly(ethylene oxide) andpoly(propylene oxide) (commercially known as Poloxamers or Poloxamines),alkyl polyglucosides, including octyl glucoside, decyl maltoside, fattyalcohols (e.g., cetyl alcohol and oleyl alcohol), cocamide MEA, cocamideDEA, polysorbates (Tween 20, Tween 80, etc.), Triton detergents, anddodecyl dimethylamine oxide.

As used herein, the term “therapeutically effective amount or dose” or“sufficient/effective amount or dose,” refers to a dose that produceseffects for which it is administered. The exact dose will depend on thepurpose of the treatment, and will be ascertainable by one skilled inthe art using known techniques (see, e.g., Lieberman, PharmaceuticalDosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technologyof Pharmaceutical Compounding (1999); Pickar, Dosage Calculations(1999); and Remington: The Science and Practice of Pharmacy, 20thEdition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins; thedisclosures of which are herein incorporated by reference in theirentireties for all purposes).

As used in this application, the term “spraying” refers to a means ofdelivering a liquid substance into a system, e.g., during an alcoholprecipitation step, such as a Cohn fractionation I or II+IIIprecipitation step, in the form of fine droplets or mist of the liquidsubstance. Spraying may be achieved by any pressurized device, such as acontainer (e.g., a spray bottle), that has a spray head or a nozzle andis operated manually or automatically to generate a fine mist from aliquid. Typically, spraying is performed while the system receiving theliquid substance is continuously stirred or otherwise mixed to ensurerapid and equal distribution of the liquid within the system.

As used herein, the term “about” denotes an approximate range of plus orminus 10% from a specified value. For instance, the language “about 20%”encompasses a range of 18-22%. As used herein, about also includes theexact amount. Hence “about 20%” means “about 20%” and also “20%.” Asused herein, “about” refers to a range of at or about the specifiedvalue.

The terms “dose” and “dosage” are used interchangeably herein. A doserefers to the amount of active ingredient given to an individual at eachadministration. The dose will vary depending on a number of factors,including frequency of administration; size and tolerance of theindividual; severity of the condition; risk of side effects; and theroute of administration. One of skill in the art will recognize that thedose can be modified depending on the above factors or based ontherapeutic progress. The term “dosage form” refers to the particularformat of the pharmaceutical, and depends on the route ofadministration. For example, a dosage form can be in a liquid, e.g., asaline solution for injection.

As used herein, the term “prevent” refers to a decreased likelihood orreduced frequency of symptoms arising from a condition associated withthe lack of function or disfunction of a blood protein.

As used herein, the term “therapy,” “treatment,” and “amelioration”refer to any reduction in the severity of symptoms arising from acondition associated with the lack of function or disfunction of a bloodprotein. As used herein, the terms “treat” and “prevent” are notintended to be absolute terms. Treatment can refer to any delay inonset, amelioration of symptoms, improvement in patient survival,increase in survival time or rate, etc. The effect of treatment can becompared to an individual or pool of individuals not receiving thetreatment.

As used herein, the term “substantial fraction” refers to at least 10%of the population of a particular protein in a composition. For example,when referring to a substantial fraction of a serine protease in acomposition, a substantial fraction of the serine protease correspondsto at least 10% of the serine protease present in the composition. Inone embodiment, a substantial fraction refers to at least 25% of thepopulation of a particular protein in a composition. In anotherembodiment, a substantial fraction refers to at least 50% of thepopulation of a particular protein in a composition. In anotherembodiment, a substantial fraction refers to at least 75% of thepopulation of a particular protein in a composition. In yet otherembodiments, a substantial fraction refers to at least 10% of thepopulation of a particular protein in a composition, or at least 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98,99, or more of the population of a particular protein in a composition.

III. Reduction of Serine Protease and Serine Protease Zymogen Content

In a first aspect, the present invention provides a method for reducingthe amount of a serine protease or a serine protease zymogen in aplasma-derived target protein composition by binding the serine proteaseand/or serine protease zymogen to finely divided silicon dioxide (SiO₂)and separating the SiO₂ from the composition.

In one embodiment, the method comprises the steps of: (a) contacting thecomposition with finely divided silicon dioxide (SiO₂) under conditionssuitable to bind at least one serine protease or serine proteasezymogen; and (b) separating the SiO₂ from the composition to remove thebound serine protease or serine protease zymogen. In a preferredembodiment, the serine protease or serine protease zymogen is Factor XIa(FXIa), Factor XIIa (FXIIa), Factor XI (FXI), and/or Factor XII (FXII).

Accordingly, in one embodiment, the invention provides a method forreducing the amount of Factor XI in a plasma-derived proteincomposition, the method comprising the steps of: (a) contacting thecomposition with finely divided silicon dioxide (SiO₂) under conditionssuitable to bind Factor XI; and (b) separating the SiO₂ from thecomposition to remove the bound Factor XI.

In another embodiment, the invention provides a method for reducing theamount of Factor XIa in a plasma-derived protein composition, the methodcomprising the steps of: (a) contacting the composition with finelydivided silicon dioxide (SiO₂) under conditions suitable to bind FactorXIa; and (b) separating the SiO₂ from the composition to remove thebound Factor XIa.

In another embodiment, the invention provides a method for reducing theamount of Factor XII in a plasma-derived protein composition, the methodcomprising the steps of: (a) contacting the composition with finelydivided silicon dioxide (SiO₂) under conditions suitable to bind FactorXII; and (b) separating the SiO₂ from the composition to remove thebound Factor XII.

In yet another embodiment, the invention provides a method for reducingthe amount of Factor XIIa in a plasma-derived protein composition, themethod comprising the steps of: (a) contacting the composition withfinely divided silicon dioxide (SiO₂) under conditions suitable to bindFactor XIIa; and (b) separating the SiO₂ from the composition to removethe bound Factor XIIa.

In certain embodiments, the method described above further comprises thestep of performing a first target protein enrichment step to form afirst enriched composition, prior to contacting the composition withfinely divided silicon dioxide (SiO₂). In certain embodiments, the firsttarget protein enrichment step is selected from a protein precipitationstep (e.g., an alcohol fractionation step), anultrafiltration/diafiltration step, and a chromatographic step.

Accordingly, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived target protein the method comprises the steps of: (a)forming a first enriched plasma-derived target protein composition bypartially precipitating protein in a starting material derived frompooled plasma; (b) contacting the first enriched composition with finelydivided silicon dioxide (SiO₂) under conditions suitable to bind atleast one serine protease or serine protease zymogen; and (c) separatingthe SiO₂ from the composition to remove the bound serine protease orserine protease zymogen. In one embodiment, the partial precipitation isachieved using alcohol. In a preferred embodiment, the alcohol isethanol. In another preferred embodiment, the serine protease or serineprotease zymogen is Factor XIa (FXIa), Factor XIIa (FXIIa), Factor XI(FXI), and/or Factor XII (FXII).

In another embodiment, the invention provides a method for reducing theamount of a serine protease or a serine protease zymogen in aplasma-derived target protein the method comprises the steps of: (a)forming a first enriched plasma-derived target protein composition byultrafiltering and/or diafiltering a starting material derived frompooled plasma; (b) contacting the first enriched composition with finelydivided silicon dioxide (SiO₂) under conditions suitable to bind atleast one serine protease or serine protease zymogen; and (c) separatingthe SiO₂ from the composition to remove the bound serine protease orserine protease zymogen. In a preferred embodiment, the serine proteaseor serine protease zymogen is Factor XIa (FXIa), Factor XIIa (FXIIa),Factor XI (FXI), and/or Factor XII (FXII).

In yet another embodiment, the invention provides a method for reducingthe amount of a serine protease or a serine protease zymogen in aplasma-derived target protein the method comprises the steps of: (a)forming a first enriched plasma-derived target protein composition bycontacting a starting material derived from pooled plasma with achromatographic resin; (b) contacting the first enriched compositionwith finely divided silicon dioxide (SiO₂) under conditions suitable tobind at least one serine protease or serine protease zymogen; and (c)separating the SiO₂ from the composition to remove the bound serineprotease or serine protease zymogen. In certain embodiments, thechromatographic resin is selected from an anion exchange resin, a cationexchange resin, a hydrophobic interaction resin, a mixed mode resin, ahydroxyapatite resin, a ligand affinity resin, an immunoaffinity resin,and a size exclusion resin. In a preferred embodiment, the serineprotease or serine protease zymogen is Factor XIa (FXIa), Factor XIIa(FXIIa), Factor XI (FXI), and/or Factor XII (FXII).

In certain embodiments, the methods described above further comprisesthe step of performing a second target protein enrichment step to form asecond enriched composition, prior to contacting the composition withfinely divided silicon dioxide (SiO₂). In certain embodiments, the firsttarget protein enrichment step is selected from a protein precipitationstep (e.g., an alcohol fractionation step), anultrafiltration/diafiltration step, and a chromatographic step.

Accordingly, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived target protein the method comprises the steps of: (a)performing a first target protein enrichment step to form a firstenriched plasma-derived target protein composition; (b) performing asecond target protein enrichment step to form a second enrichedplasma-derived target protein composition; (c) contacting the secondenriched composition with finely divided silicon dioxide (SiO₂) underconditions suitable to bind at least one serine protease or serineprotease zymogen; and (d) separating the SiO₂ from the composition toremove the bound serine protease or serine protease zymogen. In apreferred embodiment, the serine protease or serine protease zymogen isFactor XIa (FXIa), Factor XIIa (FXIIa), Factor XI (FXI), and/or FactorXII (FXII). In certain embodiments, the combination of first and secondenrichment steps is selected from any one of variations Var. 1 to Var.100, found in Table 1.

TABLE 1 Exemplary embodiments for the combination of first and secondenrichment steps. First Enrichment Step* Ppt UF/DF AEC CEC HIC HAC MMCLAC IAC SEC Second Ppt Var. 1 Var. 11 Var. 21 Var. 31 Var. 41 Var. 51Var. 61 Var. 71 Var. 81 Var. 91 Enrichment UF/DF Var. 2 Var. 12 Var. 22Var. 32 Var. 42 Var. 52 Var. 62 Var. 72 Var. 82 Var. 92 Step AEC Var. 3Var. 13 Var. 23 Var. 33 Var. 43 Var. 53 Var. 63 Var. 73 Var. 83 Var. 93CEC Var. 4 Var. 14 Var. 24 Var. 34 Var. 44 Var. 54 Var. 64 Var. 74 Var.84 Var. 94 HIC Var. 5 Var. 15 Var. 25 Var. 35 Var. 45 Var. 55 Var. 65Var. 75 Var. 85 Var. 95 HAC Var. 6 Var. 16 Var. 26 Var. 36 Var. 46 Var.56 Var. 66 Var. 76 Var. 86 Var. 96 MMC Var. 7 Var. 17 Var. 27 Var. 37Var. 47 Var. 57 Var. 67 Var. 77 Var. 87 Var. 97 LAC Var. 8 Var. 18 Var.28 Var. 38 Var. 48 Var. 58 Var. 68 Var. 78 Var. 88 Var. 98 IAC Var. 9Var. 19 Var. 29 Var. 39 Var. 49 Var. 59 Var. 69 Var. 79 Var. 89 Var. 99SEC  Var. 10 Var. 20 Var. 30 Var. 40 Var. 50 Var. 60 Var. 70 Var. 80Var. 90  Var. 100 *Ppt: Precipitation UF/DF:Ultrafiltration/Diafiltration AEC: Anion Exchange Chromatography CEC:Cation Exchange Chromatography HIC: Hydrophobic InteractionChromatography HAC: Hydroxyapatite Chromatography MMC: Mixed ModeChromatography LAC: Ligand Affinity Chromatography IAC: Immuno-AffinityChromatography SEC: Size Exclusion Chromatography

In certain embodiments, the methods described above further comprisesthe step of performing a target protein enrichment step after contactingthe composition with finely divided silicon dioxide (SiO₂). In certainembodiments, the target protein enrichment step is selected from aprotein precipitation step (e.g., an alcohol fractionation step), anultrafiltration/diafiltration step, and a chromatographic step.

Accordingly, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived target protein the method comprises the steps of: (a)performing a first target protein enrichment step to form a firstenriched plasma-derived target protein composition; (b) contacting thefirst enriched composition with finely divided silicon dioxide (SiO₂)under conditions suitable to bind at least one serine protease or serineprotease zymogen; (c) separating the SiO₂ from the composition to removethe bound serine protease or serine protease zymogen; and (d) performinga second target protein enrichment step to form a second enrichedplasma-derived target protein composition. In a preferred embodiment,the serine protease or serine protease zymogen is Factor XIa (FXIa),Factor XIIa (FXIIa), Factor XI (FXI), and/or Factor XII (FXII). Incertain embodiments, the combination of first and second enrichmentsteps is selected from any one of variations Var. 1 to Var. 100, foundin Table 1.

Likewise, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived target protein the method comprises the steps of: (a)performing a first target protein enrichment step to form a firstenriched plasma-derived target protein composition; (b) performing asecond target protein enrichment step to form a second enrichedplasma-derived target protein composition; (c) contacting the secondenriched composition with finely divided silicon dioxide (SiO₂) underconditions suitable to bind at least one serine protease or serineprotease zymogen; (d) separating the SiO₂ from the composition to removethe bound serine protease or serine protease zymogen; and (e) performinga third target protein enrichment step to form a third enrichedplasma-derived target protein composition. In a preferred embodiment,the serine protease or serine protease zymogen is Factor XIa (FXIa),Factor XIIa (FXIIa), Factor XI (FXI), and/or Factor XII (FXII). Incertain embodiments, the combination of first and second enrichmentsteps is selected from any one of variations Var. 101 to Var. 1100,found in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8,Table 9, Table 10, or Table 11.

TABLE 2 Exemplary embodiments for the combination of a firstprecipitation enrichment step, a second, and a third enrichment step.Second Enrichment Step* Ppt UF/DF AEC CEC HIC HAC MMC LAC IAC SEC ThirdPpt Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Enrichment 101 111121 131 141 151 161 171 181 191 Step UF/DF Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 102 112 122 132 142 152 162 172 182 192 AEC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 103 113 123 133 143 153 163173 183 193 CEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 104114 124 134 144 154 164 174 184 194 HIC Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 105 115 125 135 145 155 165 175 185 195 HAC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 106 116 126 136 146 156 166176 186 196 MMC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 107117 127 137 147 157 167 177 187 197 LAC Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 108 118 128 138 148 158 168 178 188 198 IAC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 109 119 129 139 149 159 169179 189 199 SEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 110120 130 140 150 160 170 180 190 200 *As per Table 1.

TABLE 3 Exemplary embodiments for the combination of a firstUltrafiltration/Diafiltration step, a second, and a third enrichmentstep. Second Enrichment Step* Ppt UF/DF AEC CEC HIC HAC MMC LAC IAC SECThird Ppt Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Enrichment201 211 221 231 241 251 261 271 281 291 Step UF/DF Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 202 212 222 232 242 252 262 272 282 292AEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 203 213 223 233243 253 263 273 283 293 CEC Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 204 214 224 234 244 254 264 274 284 294 HIC Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 205 215 225 235 245 255 265 275 285 295HAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 206 216 226 236246 256 266 276 286 296 MMC Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 207 217 227 237 247 257 267 277 287 297 LAC Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 208 218 228 238 248 258 268 278 288 298IAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 209 219 229 239249 259 269 279 289 299 SEC Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 210 220 230 240 250 260 270 280 290 300 *As per Table 1.

TABLE 4 Exemplary embodiments for the combination of a first AnionExchange Chromatography step, a second, and a third enrichment step.Second Enrichment Step* Ppt UF/DF AEC CEC HIC HAC MMC LAC IAC SEC ThirdPpt Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Enrichment 301 311321 331 341 351 361 371 381 391 Step UF/DF Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 302 312 322 332 342 352 362 372 382 392 AEC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 303 313 323 333 343 353 363373 383 393 CEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 304314 324 334 344 354 364 374 384 394 HIC Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 305 315 325 335 345 355 365 375 385 395 HAC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 306 316 326 336 346 356 366376 386 396 MMC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 307317 327 337 347 357 367 377 387 397 LAC Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 308 318 328 338 348 358 368 378 388 398 IAC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 309 319 329 339 349 359 369379 389 399 SEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 310320 330 340 350 360 370 380 390 400 *As per Table 1.

TABLE 5 Exemplary embodiments for the combination of a first CationExchange Chromatography step, a second, and a third enrichment step.Second Enrichment Step* Ppt UF/DF AEC CEC HIC HAC MMC LAC IAC SEC ThirdPpt Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Enrichment 401 411421 431 441 451 461 471 481 491 Step UF/DF Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 402 412 422 432 442 452 462 472 482 492 AEC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 403 413 423 433 443 453 463473 483 493 CEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 404414 424 434 444 454 464 474 484 494 HIC Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 405 415 425 435 445 455 465 475 485 495 HAC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 406 416 426 436 446 456 466476 486 496 MMC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 407417 427 437 447 457 467 477 487 497 LAC Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 408 418 428 438 448 458 468 478 488 498 IAC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 409 419 429 439 449 459 469479 489 499 SEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 410420 430 440 450 460 470 480 490 500 *As per Table 1.

TABLE 6 Exemplary embodiments for the combination of a first HydrophobicInteraction Chromatography step, a second, and a third enrichment step.Second Enrichment Step* Ppt UF/DF AEC CEC HIC HAC MMC LAC IAC SEC ThirdPpt Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Enrichment 501 511521 531 541 551 561 571 581 591 Step UF/DF Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 502 512 522 532 542 552 562 572 582 592 AEC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 503 513 523 533 543 553 563573 583 593 CEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 504514 524 534 544 554 564 574 584 594 HIC Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 505 515 525 535 545 555 565 575 585 595 HAC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 506 516 526 536 546 556 566576 586 596 MMC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 507517 527 537 547 557 567 577 587 597 LAC Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 508 518 528 538 548 558 568 578 588 598 IAC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 509 519 529 539 549 559 569579 589 599 SEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 510520 530 540 550 560 570 580 590 600 *As per Table 1.

TABLE 7 Exemplary embodiments for the combination of a firstHydroxyapatite Chromatography step, a second, and a third enrichmentstep. Second Enrichment Step* Ppt UF/DF AEC CEC HIC HAC MMC LAC IAC SECThird Ppt Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Enrichment601 611 621 631 641 651 661 671 681 691 Step UF/DF Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 602 612 622 632 642 652 662 672 682 692AEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 603 613 623 633643 653 663 673 683 693 CEC Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 604 614 624 634 644 654 664 674 684 694 HIC Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 605 615 625 635 645 655 665 675 685 695HAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 606 616 626 636646 656 666 676 686 696 MMC Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 607 617 627 637 647 657 667 677 687 697 LAC Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 608 618 628 638 648 658 668 678 688 698IAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 609 619 629 639649 659 669 679 689 699 SEC Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 610 620 630 640 650 660 670 680 690 700 *As per Table 1.

TABLE 8 Exemplary embodiments for the combination of a first Mixed ModeChromatography step, a second, and a third enrichment step. SecondEnrichment Step* Ppt UF/DF AEC CEC HIC HAC MMC LAC IAC SEC Third PptVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Enrichment 701 711 721731 741 751 761 771 781 791 Step UF/DF Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 702 712 722 732 742 752 762 772 782 792 AEC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 703 713 723 733 743 753 763773 783 793 CEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 704714 724 734 744 754 764 774 784 794 HIC Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 705 715 725 735 745 755 765 775 785 795 HAC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 706 716 726 736 746 756 766776 786 796 MMC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 707717 727 737 747 757 767 777 787 797 LAC Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 708 718 728 738 748 758 768 778 788 798 IAC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 709 719 729 739 749 759 769779 789 799 SEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 710720 730 740 750 760 770 780 790 800 *As per Table 1.

TABLE 9 Exemplary embodiments for the combination of a first LigandAffinity Chromatography step, a second, and a third enrichment step.Second Enrichment Step* Ppt UF/DF AEC CEC HIC HAC MMC LAC IAC SEC ThirdPpt Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Enrichment 801 811821 831 841 851 861 871 881 891 Step UF/DF Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 802 812 822 832 842 852 862 872 882 892 AEC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 803 813 823 833 843 853 863873 883 893 CEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 804814 824 834 844 854 864 874 884 894 HIC Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 805 815 825 835 845 855 865 875 885 895 HAC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 806 816 826 836 846 856 866876 886 896 MMC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 807817 827 837 847 857 867 877 887 897 LAC Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 808 818 828 838 848 858 868 878 888 898 IAC Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 809 819 829 839 849 859 869879 889 899 SEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 810820 830 840 850 860 870 880 890 900 *As per Table 1.

TABLE 10 Exemplary embodiments for the combination of a firstImmuno-Affinity Chromatography step, a second, and a third enrichmentstep. Second Enrichment Step* Ppt UF/DF AEC CEC HIC HAC MMC LAC IAC SECThird Ppt Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Enrichment901 911 921 931 941 951 961 971 981 991 Step UF/DF Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 902 912 922 932 942 952 962 972 982 992AEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 903 913 923 933943 953 963 973 983 993 CEC Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 904 914 924 934 944 954 964 974 984 994 HIC Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 905 915 925 935 945 955 965 975 985 995HAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 906 916 926 936946 956 966 976 986 996 MMC Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 907 917 927 937 947 957 967 977 987 997 LAC Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 908 918 928 938 948 958 968 978 988 998IAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 909 919 929 939949 959 969 979 989 999 SEC Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 910 920 930 940 950 960 970 980 990 1000  *As per Table 1.

TABLE 11 Exemplary embodiments for the combination of a first SizeExclusion Chromatography step, a second, and a third enrichment step.Second Enrichment Step* Ppt UF/DF AEC CEC HIC HAC MMC LAC IAC SEC ThirdPpt Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Enrichment 10011011 1021 1031 1041 1051 1061 1071 1081 1091 Step UF/DF Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. 1002 1012 1022 1032 1042 1052 10621072 1082 1092 AEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.1003 1013 1023 1033 1043 1053 1063 1073 1083 1093 CEC Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. 1004 1014 1024 1034 1044 1054 10641074 1084 1094 HIC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.1005 1015 1025 1035 1045 1055 1065 1075 1085 1095 HAC Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. 1006 1016 1026 1036 1046 1056 10661076 1086 1096 MMC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.1007 1017 1027 1037 1047 1057 1067 1077 1087 1097 LAC Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. 1008 1018 1028 1038 1048 1058 10681078 1088 1098 IAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.1009 1019 1029 1039 1049 1059 1069 1079 1089 1099 SEC Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. 1010 1020 1030 1040 1050 1060 10701080 1090 1100 *As per Table 1.

In certain embodiments of the methods described above, a chromatographicenrichment step comprises the sub-steps of: (i) contacting theplasma-derived target protein composition with a chromatographic resinunder conditions suitable to bind the plasma-derived target protein; and(ii) eluting the plasma-derived target protein from the chromatographicresin. In one specific embodiment, the impurity does not bind to thechromatographic resin in sub-step (i). In another specific embodiment,the impurity binds to the chromatographic resin in sub-step (i), but isnot eluted from the chromatographic resin in sub-step (ii).

In other certain embodiments of the methods described above, achromatographic enrichment step comprises the sub-steps of: (i)contacting the first enriched plasma-derived target protein compositionwith a chromatographic resin under conditions suitable to bind at leastone impurity; and (ii) separating the resin from the plasma-derivedprotein composition, wherein the plasma-derived target protein does notbind to the chromatographic resin in sub-step (i).

In certain embodiments of the methods described above, theplasma-derived target protein is selected from an immunoglobulin (Ig),albumin, alpha-1-antitrypsin (A1PI), butyrylcholinesterase, a protein ofthe complement system (e.g., Factor H), and an inter-alpha-trypsininhibitor (IαI). In a specific embodiment, the protein of the complementsystem is selected from the group consisting of Factor H (FH), Factor D,complement protein C3, and C4 binding protein. In a preferredembodiment, the protein composition is a manufacturing intermediate.

In certain embodiments of the methods provided herein, the amount of aparticular serine protease or serine protease zymogen is reduced by atleast 10%. In another embodiment, the amount of a particular serineprotease or serine protease zymogen is reduced by at least 25%. Inanother embodiment, the amount of a particular serine protease or serineprotease zymogen is reduced by at least 50%. In another embodiment, theamount of a particular serine protease or serine protease zymogen isreduced by at least 75%. In another embodiment, the amount of aparticular serine protease or serine protease zymogen is reduced by atleast 90%. In yet other embodiments, the amount of a particular serineprotease or serine protease zymogen is reduced by at least 5%, or by atleast 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or tolevels below the detection limit of the test system.

Generally, the amount of finely divided silicon dioxide (SiO₂) requiredfor the methods described herein will vary dependent on several factors,including without limitation, the total amount of protein present in thecomposition, the concentration of serine protease and serine proteasezymogen (e.g., FXI, FXIa, FXII, and FXIIa) in the composition, thetarget protein, and the solution conditions (e.g., pH, conductivity,etc.). For example, SiO₂ may be added to a target composition at aconcentration between about 0.01 g/g protein and about 10 g/g protein.In another embodiment, SiO₂ may be added to a target composition at aconcentration between about 1 g/g protein and about 5 g/g protein. Inanother embodiment, SiO₂ may be added to a target composition at aconcentration between about 2 g/g protein and about 4 g/g protein. Inone embodiment, SiO₂ is added at a final concentration of at least 1 gper gram total protein. In another specific embodiment, fumed silica isadded at a concentration of at least 2 g per gram total protein. Inanother specific embodiment, fumed silica is added at a concentration ofat least 2.5 g per gram total protein. In another embodiment, SiO₂ maybe added to a target composition at a concentration between about 0.01g/g protein and about 5 g/g protein. In another embodiment, SiO₂ may beadded to a target composition at a concentration between about 0.02 g/gprotein and about 4 g/g protein. In one embodiment, SiO₂ is added at afinal concentration of at least 0.1 g per gram total protein. In anotherspecific embodiment, fumed silica is added at a concentration of atleast 0.2 g per gram total protein. In another specific embodiment,fumed silica is added at a concentration of at least 0.25 g per gramtotal protein. In yet other specific embodiments, finely divided silicondioxide is added at a concentration of at least 0.01 g/g total proteinor at least 0.02 g, 0.03 g, 0.04 g, 0.05 g, 0.06 g, 0.07 g, 0.08 g, 0.09g, 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1.0 g,1.5 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g, 4.0 g, 4.5 g, 5.0 g, 5.5 g, 6.0 g,6.5 g, 7.0 g, 7.5 g, 8.0 g, 8.5 g, 9.0 g, 9.5 g, 10.0 g, or more g/gtotal protein.

In certain embodiments in which a target protein is extracted from asuspended plasma precipitate fraction, filter aid, for example CelpureC300 (Celpure) or Hyflo-Supper-Cel (World Minerals), will be added afterthe silica dioxide treatment, to facilitate depth filtration. Filter aidcan be added at a final concentration of from about 0.01 kg/kgprecipitate to about 1.0 kg/kg precipitate, or from about 0.02 kg/kgprecipitate to about 0.8 kg/kg precipitate, or from about 0.03 kg/kgprecipitate to about 0.7 kg/kg precipitate. In other embodiments, filteraid can be added at a final concentration of from about 0.01 kg/kgprecipitate to about 0.07 kg/kg precipitate, or from about 0.02 kg/kgprecipitate to about 0.06 kg/kg precipitate, or from about 0.03 kg/kgprecipitate to about 0.05 kg/kg precipitate. In certain embodiments, thefilter aid will be added at a final concentration of about 0.01 kg/kgprecipitate, or about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 kg/kg precipitate.

A. Immunoglobulins

In one embodiment, the present invention provides a method for reducingthe amount of a serine protease or a serine protease zymogen in aplasma-derived immunoglobulin (Ig) composition. In one specificembodiment, the method comprises the steps of: (a) contacting the Igcomposition with finely divided silicon dioxide (SiO₂) under conditionssuitable to bind at least one serine protease or serine proteasezymogen; and (b) separating the SiO₂ from the Ig composition to removethe bound serine protease or serine protease zymogen. In a preferredembodiment, the serine protease or serine protease zymogen is Factor XIa(FXIa), Factor XIIa (FXIIa), Factor XI (FXI), and/or Factor XII (FXII).In one embodiment, the Ig composition is an IgG composition. In otherembodiments, the Ig composition is an IgA, IgM, IgG, or mixedcomposition thereof.

In one embodiment, the method further comprises the step of performing afirst Ig protein enrichment step to form a first enriched Igcomposition, prior to contacting the composition with finely dividedsilicon dioxide (SiO₂). In certain embodiments, the first Ig proteinenrichment step is selected from a protein precipitation step (e.g., analcohol fractionation step), an ultrafiltration/diafiltration step, anda chromatographic step. In one embodiment, the Ig composition is an IgGcomposition. In other embodiments, the Ig composition is an IgA, IgM,IgG, or mixed composition thereof.

In certain embodiments, the methods described above further comprisesthe step of performing a second Ig protein enrichment step to form asecond enriched Ig composition, prior to contacting the composition withfinely divided silicon dioxide (SiO₂). In certain embodiments, the firstIg protein enrichment step is selected from a protein precipitation step(e.g., an alcohol fractionation step), an ultrafiltration/diafiltrationstep, and a chromatographic step.

Accordingly, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived Ig composition, the method comprises the steps of: (a)performing a first Ig enrichment step to form a first enrichedplasma-derived Ig composition; (b) performing a second Ig enrichmentstep to form a second enriched plasma-derived Ig composition; (c)contacting the second enriched composition with finely divided silicondioxide (SiO₂) under conditions suitable to bind at least one serineprotease or serine protease zymogen; and (d) separating the SiO₂ fromthe composition to remove the bound serine protease or serine proteasezymogen. In a preferred embodiment, the serine protease or serineprotease zymogen is Factor XIa (FXIa), Factor XIIa (FXIIa), Factor XI(FXI), and/or Factor XII (FXII). In certain embodiments, the combinationof first and second enrichment steps is selected from any one ofvariations Var. 1 to Var. 100, found in Table 1.

In certain embodiments, the methods described above further comprisesthe step of performing an Ig enrichment step after contacting thecomposition with finely divided silicon dioxide (SiO₂). In certainembodiments, the Ig enrichment step is selected from a proteinprecipitation step (e.g., an alcohol fractionation step), anultrafiltration/diafiltration step, and a chromatographic step.

Accordingly, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived Ig composition the method comprises the steps of: (a)performing a first Ig enrichment step to form a first enrichedplasma-derived Ig composition; (b) contacting the first enrichedcomposition with finely divided silicon dioxide (SiO₂) under conditionssuitable to bind at least one serine protease or serine proteasezymogen; (c) separating the SiO₂ from the composition to remove thebound serine protease or serine protease zymogen; and (d) performing asecond Ig enrichment step to form a second enriched plasma-derived Igcomposition. In a preferred embodiment, the serine protease or serineprotease zymogen is Factor XIa (FXIa), Factor XIIa (FXIIa), Factor XI(FXI), and/or Factor XII (FXII). In certain embodiments, the combinationof first and second enrichment steps is selected from any one ofvariations Var. 1 to Var. 100, found in Table 1.

Likewise, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived Ig composition, the method comprising the steps of: (a)performing a first Ig enrichment step to form a first enrichedplasma-derived Ig composition; (b) performing a second Ig enrichmentstep to form a second enriched plasma-derived Ig composition; (c)contacting the second enriched composition with finely divided silicondioxide (SiO₂) under conditions suitable to bind at least one serineprotease or serine protease zymogen; (d) separating the SiO₂ from thecomposition to remove the bound serine protease or serine proteasezymogen; and (e) performing a third Ig enrichment step to form a thirdenriched plasma-derived Ig composition. In a preferred embodiment, theserine protease or serine protease zymogen is Factor XIa (FXIa), FactorXIIa (FXIIa), Factor XI (FXI), and/or Factor XII (FXII). In certainembodiments, the combination of first and second enrichment steps isselected from any one of variations Var. 101 to Var. 1100, found inTable 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9,Table 10, or Table 11.

In a particular embodiment, the Ig composition is a manufacturingintermediate. For example, in certain embodiments, the Ig composition isan IgG manufacturing intermediate from a Cohn fractionation procedure(J. Am. Chem. Soc., 1946, 68(3): 459-475; J. Am. Chem. Soc. 72:465-474(1950)), an Oncley fractionation procedure (J. Am. Chem. Soc., 1949,71(2): 541-550), a Deutsch purification procedure (J. Biol. Chem.164:109-118), a Hoppe purification procedure (Munch Med Wochenschr 1967(34): 1749-1752), a Falksveden purification procedure (Swedish PatentNo. 348942), a Falksveden and Lundblad purification procedure (Methodsof Plasma Protein Fractionation 1980), a Lebing purification procedure(Vox Sang 2003 (84):193-201), a Tanaka purification procedure (Braz JMed Biol Res 2000 (33)37-30)), a Teschner purification procedure (VoxSang, 2007 (92):42-55), a Nitschmann fractionation procedure (Helv.Chim. Acta 37:866-873), a Kistler/Nitschmann fractionation procedure(Vox Sang. 7:414-424 (1962)), a Barundern purification procedure (VoxSang. 7:157-74 (1962)), a Koblet purification procedure (Vox Sang.13:93-102 (1967)) a purification procedure disclosed in U.S. Pat. No.5,122,373 or 5,177,194, modified procedures thereof, and similar orequivalent purification procedures known in the art.

In one particular embodiment, the IgG composition is a cryo-poor Cohnpool. In another particular embodiment, the IgG composition is a CohnFraction I supernatant or equivalent fraction thereof. In anotherparticular embodiment, the IgG composition is a re-suspended CohnFraction III precipitate, or equivalent fraction thereof. In anotherparticular embodiment, the IgG composition is a re-suspended CohnFraction II+III precipitate, or equivalent fraction thereof. In anotherparticular embodiment, the IgG composition is a re-suspended CohnFraction I+II+III precipitate, or equivalent fraction thereof. Inanother particular embodiment, the IgG composition is a re-suspendedPrecipitate G precipitate, or equivalent fraction thereof. In anotherparticular embodiment, the IgG composition is a re-suspendedKistler/Nitschmann Precipitate B precipitate, or equivalent fractionthereof.

In a specific embodiment, the present invention provides a method forreducing the amount of serine protease and/or serine protease zymogen ina re-suspended IgG Fraction II+III precipitate. Advantageously, it hasbeen found that the levels of Factor XI, Factor XII, Factor XIa, and/orFactor XIIa in a re-suspended IgG Fraction II+III precipitate can begreatly reduced by the addition of a pretreatment step prior tofiltration/centrifugation. In one embodiment, this pretreatment stepcomprises addition of finely divided silica dioxide particles (e.g.,fumed silica, Aerosil®) followed by a 40 to 80 minute incubation periodduring which the suspension is constantly mixed. In certain embodiments,the incubation period will be between about 50 minutes and about 70minutes, or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, ormore minutes. Generally, the treatment will be performed at betweenabout 0° C. and about 10° C., or between about 2° C. and about 8° C. Incertain embodiments, the treatment may be performed at about 0° C., 1°C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., or 10° C. Ina particular embodiment, the treatment is performed at between about 2°C. and about 10° C.

The effect of the fumed silica treatment is exemplified by the resultsfound in Examples 3, 6, and 7. In these examples, Fraction II+IIIprecipitates are re-suspended and treated with varying amounts of finelydivided silicon dioxide. As can be seen in Table 22, Table 27, Table 28,and

Table 29, Factor XI and XII serine protease activity and zymogen contentcan be reduced at least 90% by treating the suspension with SiO₂.

In certain embodiments, fumed silica is added at a concentration ofbetween about 20 g/kg II+III paste and about 100 g/kg II+III paste(i.e., for a Modified Fraction II+III precipitate that is extracted at aratio of 1:15, fumed silica should be added at a concentration fromabout 20 g/16 kg II+III suspension to about 100 g/16 kg II+IIIsuspension, or at a final concentration of about 0.125% (w/w) to about0.625% (w/w)). In certain embodiments, the fumed silica may be added ata concentration of about 20 g/kg II+III paste, or about 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 g/kg II+III paste. Inone specific embodiment, fumed silica (e.g., Aerosil 380 or equivalent)is added to the Modified Fraction II+III suspension to a finalconcentration of about 40 g/16 kg II+III. Mixing takes place at about 2to 8° C. for at least 50 to 70 minutes.

In certain embodiments, SiO₂ is added to a an IgG composition at aconcentration between about 0.01 g/g protein and about 10 g/g protein.In another embodiment, SiO₂ is added to a an IgG composition at aconcentration between about 0.01 g/g protein and about 5 g/g protein. Inanother embodiment, SiO₂ is added to an IgG composition at aconcentration between about 0.02 g/g protein and about 4 g/g protein. Inone embodiment, SiO₂ is added at a final concentration of at least 0.1 gper gram total protein. In another specific embodiment, fumed silica isadded at a concentration of at least 0.2 g per gram total protein. Inanother specific embodiment, fumed silica is added at a concentration ofat least 0.25 g per gram total protein. In other specific embodiments,fumed silica is added at a concentration of at least 1 g per gram totalprotein. In another specific embodiment, fumed silica is added at aconcentration of at least 2 g per gram total protein. In anotherspecific embodiment, fumed silica is added at a concentration of atleast 2.5 g per gram total protein. In yet other specific embodiments,finely divided silicon dioxide is added at a concentration of at least0.01 g/g total protein or at least 0.02 g, 0.03 g, 0.04 g, 0.05 g, 0.06g, 0.07 g, 0.08 g, 0.09 g, 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7g, 0.8 g, 0.9 g, 1.0 g, 1.5 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g, 4.0 g, 4.5 g,5.0 g, 5.5 g, 6.0 g, 6.5 g, 7.0 g, 7.5 g, 8.0 g, 8.5 g, 9.0 g, 9.5 g,10.0 g, or more per gram total protein.

In certain embodiments, filter aid, for example Celpure C300 (Celpure)or Hyflo-Supper-Cel (World Minerals), will be added after the silicadioxide treatment, to facilitate depth filtration. Filter aid can beadded at a final concentration of from about 0.01 kg/kg II+III paste toabout 1.0 kg/kg II+III paste, or from about 0.02 kg/kg II+III paste toabout 0.8 kg/kg II+III paste, or from about 0.03 kg/kg II+III paste toabout 0.7 kg/kg II+III paste. In other embodiments, filter aid can beadded at a final concentration of from about 0.01 kg/kg II+III paste toabout 0.07 kg/kg II+III paste, or from about 0.02 kg/kg II+III paste toabout 0.06 kg/kg II+III paste, or from about 0.03 kg/kg II+III paste toabout 0.05 kg/kg II+III paste. In certain embodiments, the filter aidwill be added at a final concentration of about 0.01 kg/kg II+III paste,or about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 kg/kg II+III paste.

In one embodiment, the process improvements are realized by inclusion ofa fumed silica treatment prior to filtration or centrifugalclarification of a Fraction II+III suspension. In certain embodiments,the fumed silica treatment will include addition of from about 0.01kg/kg II+III paste to about 0.07 kg/kg II+III paste, or from about 0.02kg/kg II+III paste to about 0.06 kg/kg II+III paste, or from about 0.03kg/kg II+III paste to about 0.05 kg/kg II+III paste, or about 0.02 kg/kgII+III paste, 0.03 kg/kg II+III paste, 0.04 kg/kg II+III paste, 0.05kg/kg II+III paste, 0.06 kg/kg II+III paste, 0.07 kg/kg II+III paste,0.08 kg/kg II+III paste, 0.09 kg/kg II+III paste, or 0.1 kg/kg II+IIIpaste, and the mixture will be incubated for between about 50 minutesand about 70 minutes, or about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, or more minutes at a temperature between about 2° C. and about 8° C.In another embodiment, the process improvements are realized byinclusion of a fumed silica treatment which reduced the levels ofresidual fibrinogen, amidolytic activity, and/or prekallikrein activatoractivity. In a specific embodiment, the process improvements arerealized by inclusion of a fumed silica treatment, which reduces thelevels of FXI, FXIa, FXII, and FXIIa in the immunoglobulin preparation.

Generally, serine protease and/or serine protease zymogen removal fromimmunoglobulin compositions can be achieved by treating theimmunoglobulin-containing solution with finely divided silicon dioxide(SiO₂) under pH and conductivity solution conditions in which the serineprotease and/or serine protease zymogen binds to the SiO₂. As shown inthe examples, suitable conditions include low pH and low conductivity.

Accordingly, in one embodiment, the present invention provides a methodfor reducing the amount of a serine protease or a serine proteasezymogen in a plasma-derived immunoglobulin composition, the methodcomprising contacting the composition with SiO₂ at a pH between about4.0 and about 7.0 to bind a serine protease or a serine protease zymogenand removing the SiO₂ from the composition. In another embodiment, themethod comprises contacting the composition with SiO₂ at a pH betweenabout 4.0 and about 6.5. In another embodiment, the method comprisescontacting the composition with SiO₂ at a pH between about 4.0 and about6.0. In another embodiment, the method comprises contacting thecomposition with SiO₂ at a pH between about 4.0 and about 5.5. Inanother embodiment, the method comprises contacting the composition withSiO₂ at a pH between about 4.0 and about 5.0. In another embodiment, themethod comprises contacting the composition with SiO₂ at a pH betweenabout 4.5 and about 7.0. In another embodiment, the method comprisescontacting the composition with SiO₂ at a pH between about 4.5 and about6.5. In another embodiment, the method comprises contacting thecomposition with SiO₂ at a pH between about 4.5 and about 6.0. Inanother embodiment, the method comprises contacting the composition withSiO₂ at a pH between about 4.5 and about 5.5. In another embodiment, themethod comprises contacting the composition with SiO₂ at a pH betweenabout 4.5 and about 5.0. In another embodiment, the method comprisescontacting the composition with SiO₂ at a pH between about 5.0 and about7.0. In another embodiment, the method comprises contacting thecomposition with SiO₂ at a pH between about 5.0 and about 6.5. Inanother embodiment, the method comprises contacting the composition withSiO₂ at a pH between about 5.0 and about 6.0. In another embodiment, themethod comprises contacting the composition with SiO₂ at a pH betweenabout 5.0 and about 5.5. In yet another embodiment, the method comprisescontacting the composition with SiO₂ at a pH between about 4.6 and about5.6. In another embodiment, the method comprises contacting thecomposition with SiO₂ at a pH between about 4.7 and about 5.5. Inanother embodiment, the method comprises contacting the composition withSiO₂ at a pH between about 4.8 and about 5.4. In another embodiment, themethod comprises contacting the composition with SiO₂ at a pH betweenabout 4.9 and about 5.3. In another embodiment, the method comprisescontacting the composition with SiO₂ at a pH between about 5.0 and about5.2. In another embodiment, the method comprises contacting thecomposition with SiO₂ at a pH of about 5.1. In other embodiments, themethod comprises contacting the composition with SiO₂ at a pH of about4.0 or about 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, or no more than 7.0. In yet other embodiments, the methodcomprises contacting the composition with SiO₂ at a pH of no more than4.0 or no more than 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, or no more than 7.0.

In one embodiment, the present invention provides a method for reducingthe amount of a serine protease or a serine protease zymogen in aplasma-derived immunoglobulin composition, the method comprisingcontacting the composition with SiO₂ at an ionic strength between about0.1 mS/cm and about 3.0 mS/cm to bind a serine protease or a serineprotease zymogen and removing the SiO₂ from the composition. In anotherembodiment, the method comprises contacting the composition with SiO₂ atan ionic strength between about 0.5 mS/cm and about 2.0 mS/cm. Inanother embodiment, the method comprises contacting the composition withSiO₂ at an ionic strength between about 1.3 mS/cm and about 1.7 mS/cm.In another embodiment, the method comprises contacting the compositionwith SiO₂ at an ionic strength between about 0.1 mS/cm and about 1.9mS/cm. In another embodiment, the method comprises contacting thecomposition with SiO₂ at an ionic strength between about 0.1 mS/cm andabout 1.8 mS/cm. In another embodiment, the method comprises contactingthe composition with SiO₂ at an ionic strength between about 0.1 mS/cmand about 1.7 mS/cm. In another embodiment, the method comprisescontacting the composition with SiO₂ at an ionic strength between about0.1 mS/cm and about 1.6 mS/cm. In another embodiment, the methodcomprises contacting the composition with SiO₂ at an ionic strengthbetween about 0.1 mS/cm and about 1.5 mS/cm. In another embodiment, themethod comprises contacting the composition with SiO₂ at an ionicstrength between about 0.1 mS/cm and about 1.4 mS/cm. In anotherembodiment, the method comprises contacting the composition with SiO₂ atan ionic strength between about 0.1 mS/cm and about 1.3 mS/cm. Inanother embodiment, the method comprises contacting the composition withSiO₂ at an ionic strength between about 0.1 mS/cm and about 1.2 mS/cm.In another embodiment, the method comprises contacting the compositionwith SiO₂ at an ionic strength between about 0.1 mS/cm and about 1.1mS/cm. In another embodiment, the method comprises contacting thecomposition with SiO₂ at an ionic strength between about 0.1 mS/cm andabout 1.0 mS/cm. In another embodiment, the method comprises contactingthe composition with SiO₂ at an ionic strength between about 0.1 mS/cmand about 0.9 mS/cm. In another embodiment, the method comprisescontacting the composition with SiO₂ at an ionic strength between about0.1 mS/cm and about 0.8 mS/cm. In another embodiment, the methodcomprises contacting the composition with SiO₂ at an ionic strengthbetween about 0.2 mS/cm and about 1.0 mS/cm. In another embodiment, themethod comprises contacting the composition with SiO₂ at an ionicstrength between about 0.3 mS/cm and about 1.0 mS/cm. In anotherembodiment, the method comprises contacting the composition with SiO₂ atan ionic strength between about 0.1 mS/cm and about 0.4 mS/cm. Inanother embodiment, the method comprises contacting the composition withSiO₂ at an ionic strength between about 0.5 mS/cm and about 1.0 mS/cm.In another embodiment, the method comprises contacting the compositionwith SiO₂ at an ionic strength between about 0.6 mS/cm and about 1.0mS/cm. In another embodiment, the method comprises contacting thecomposition with SiO₂ at an ionic strength between about 0.7 mS/cm andabout 0.9 mS/cm. In another embodiment, the method comprises contactingthe composition with SiO₂ at an ionic strength of about 0.8 mS/cm. Inother embodiments, the method comprises contacting the composition withSiO₂ at an ionic strength of about 0.1 mS/cm or no more than 0.2 mS/cm,0.3 mS/cm, 0.4 mS/cm, 0.5 mS/cm, 0.6 mS/cm, 0.7 mS/cm, 0.8 mS/cm, 0.9mS/cm, 1.0 mS/cm, 1.1 mS/cm, 1.2 mS/cm, 1.3 mS/cm, 1.4 mS/cm, 1.5 mS/cm,1.6 mS/cm, 1.7 mS/cm, 1.8 mS/cm, 1.9 mS/cm, 2.0 mS/cm, 2.1 mS/cm, 2.2mS/cm, 2.3 mS/cm, 2.4 mS/cm, 2.5 mS/cm, 2.6 mS/cm, 2.7 mS/cm, 2.8 mS/cm,2.9 mS/cm, or 3.0 mS/cm. In yet other embodiments, the method comprisescontacting the composition with SiO₂ at an ionic strength of no morethan 0.1 mS/cm or no more than 0.2 mS/cm, 0.3 mS/cm, 0.4 mS/cm, 0.5mS/cm, 0.6 mS/cm, 0.7 mS/cm, 0.8 mS/cm, 0.9 mS/cm, 1.0 mS/cm, 1.1 mS/cm,1.2 mS/cm, 1.3 mS/cm, 1.4 mS/cm, 1.5 mS/cm, 1.6 mS/cm, 1.7 mS/cm, 1.8mS/cm, 1.9 mS/cm, 2.0 mS/cm, 2.1 mS/cm, 2.2 mS/cm, 2.3 mS/cm, 2.4 mS/cm,2.5 mS/cm, 2.6 mS/cm, 2.7 mS/cm, 2.8 mS/cm, 2.9 mS/cm, or 3.0 mS/cm.

In certain embodiments, the present invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived immunoglobulin composition, the method comprisingcontacting the composition with SiO₂ at a low pH and low ionic strengthto bind a serine protease or a serine protease zymogen and removing theSiO₂ from the composition. In a particular embodiment, the methodcomprises contacting the composition with SiO₂ at a pH between about 4.8and about 5.4 at an ionic strength between about 0.6 mS/cm and about 1.0mS/cm. In a more particular embodiment, the method comprises contactingthe composition with SiO₂ at a pH between about 4.9 and about 5.3 at anionic strength between about 0.7 mS/cm and about 0.9 mS/cm. In a yetmore particular embodiment, the method comprises contacting thecomposition with SiO₂ at a pH between about 5.0 and about 5.2 at anionic strength of about 0.8 mS/cm. In yet other embodiments, the methodcomprises contacting the composition with SiO₂ at a pH and ionicstrength according to any one of variations Var. 1222 to 3041, aspresented in Table 12, Table 13, Table 14, and Table 15.

TABLE 12 Exemplary embodiments of solution conditions useful for bindingserine proteases and/or serine protease zymogens to SiO₂. pH 4.0-7.04.5-5.0 4.5-5.0 4.5-5.0 4.5-5.0 4.5-5.0 4.5-5.0 4.5-5.0 4.5-5.0 Ionic0.1-2.0 Var. 1222 Var. 1638 Var. 1638 Var. 1638 Var. 1638 Var. 1638 Var.1638 Var. 1638 Var. 1638 Strength 0.1-1.9 Var. 1223 Var. 1639 Var. 1639Var. 1639 Var. 1639 Var. 1639 Var. 1639 Var. 1639 Var. 1639 (mS/cm)0.1-1.8 Var. 1224 Var. 1640 Var. 1640 Var. 1640 Var. 1640 Var. 1640 Var.1640 Var. 1640 Var. 1640 0.1-1.7 Var. 1225 Var. 1641 Var. 1641 Var. 1641Var. 1641 Var. 1641 Var. 1641 Var. 1641 Var. 1641 0.1-1.6 Var. 1226 Var.1642 Var. 1642 Var. 1642 Var. 1642 Var. 1642 Var. 1642 Var. 1642 Var.1642 0.1-1.5 Var. 1227 Var. 1643 Var. 1643 Var. 1643 Var. 1643 Var. 1643Var. 1643 Var. 1643 Var. 1643 0.1-1.4 Var. 1228 Var. 1644 Var. 1644 Var.1644 Var. 1644 Var. 1644 Var. 1644 Var. 1644 Var. 1644 0.1-1.3 Var. 1229Var. 1645 Var. 1645 Var. 1645 Var. 1645 Var. 1645 Var. 1645 Var. 1645Var. 1645 0.1-1.2 Var. 1230 Var. 1646 Var. 1646 Var. 1646 Var. 1646 Var.1646 Var. 1646 Var. 1646 Var. 1646 0.1-1.1 Var. 1231 Var. 1647 Var. 1647Var. 1647 Var. 1647 Var. 1647 Var. 1647 Var. 1647 Var. 1647 0.1-1.0 Var.1232 Var. 1648 Var. 1648 Var. 1648 Var. 1648 Var. 1648 Var. 1648 Var.1648 Var. 1648 0.1-0.9 Var. 1233 Var. 1649 Var. 1649 Var. 1649 Var. 1649Var. 1649 Var. 1649 Var. 1649 Var. 1649 0.1-0.8 Var. 1234 Var. 1650 Var.1650 Var. 1650 Var. 1650 Var. 1650 Var. 1650 Var. 1650 Var. 1650 0.2-2.0Var. 1235 Var. 1651 Var. 1651 Var. 1651 Var. 1651 Var. 1651 Var. 1651Var. 1651 Var. 1651 0.2-1.5 Var. 1236 Var. 1652 Var. 1652 Var. 1652 Var.1652 Var. 1652 Var. 1652 Var. 1652 Var. 1652 0.2-1.0 Var. 1237 Var. 1653Var. 1653 Var. 1653 Var. 1653 Var. 1653 Var. 1653 Var. 1653 Var. 16530.2-0.9 Var. 1238 Var. 1654 Var. 1654 Var. 1654 Var. 1654 Var. 1654 Var.1654 Var. 1654 Var. 1654 0.2-0.8 Var. 1239 Var. 1655 Var. 1655 Var. 1655Var. 1655 Var. 1655 Var. 1655 Var. 1655 Var. 1655 0.3-1.0 Var. 1240 Var.1656 Var. 1656 Var. 1656 Var. 1656 Var. 1656 Var. 1656 Var. 1656 Var.1656 0.3-0.9 Var. 1241 Var. 1657 Var. 1657 Var. 1657 Var. 1657 Var. 1657Var. 1657 Var. 1657 Var. 1657 0.3-0.8 Var. 1242 Var. 1658 Var. 1658 Var.1658 Var. 1658 Var. 1658 Var. 1658 Var. 1658 Var. 1658 0.4-1.0 Var. 1243Var. 1659 Var. 1659 Var. 1659 Var. 1659 Var. 1659 Var. 1659 Var. 1659Var. 1659 0.4-0.9 Var. 1244 Var. 1660 Var. 1660 Var. 1660 Var. 1660 Var.1660 Var. 1660 Var. 1660 Var. 1660 0.4-0.8 Var. 1245 Var. 1661 Var. 1661Var. 1661 Var. 1661 Var. 1661 Var. 1661 Var. 1661 Var. 1661 0.5-1.0 Var.1246 Var. 1662 Var. 1662 Var. 1662 Var. 1662 Var. 1662 Var. 1662 Var.1662 Var. 1662 0.5-0.9 Var. 1247 Var. 1663 Var. 1663 Var. 1663 Var. 1663Var. 1663 Var. 1663 Var. 1663 Var. 1663 0.5-0.8 Var. 1248 Var. 1664 Var.1664 Var. 1664 Var. 1664 Var. 1664 Var. 1664 Var. 1664 Var. 1664 0.6-1.0Var. 1249 Var. 1665 Var. 1665 Var. 1665 Var. 1665 Var. 1665 Var. 1665Var. 1665 Var. 1665 0.6-0.9 Var. 1250 Var. 1666 Var. 1666 Var. 1666 Var.1666 Var. 1666 Var. 1666 Var. 1666 Var. 1666 0.6-0.8 Var. 1251 Var. 1667Var. 1667 Var. 1667 Var. 1667 Var. 1667 Var. 1667 Var. 1667 Var. 16670.7-1.0 Var. 1252 Var. 1668 Var. 1668 Var. 1668 Var. 1668 Var. 1668 Var.1668 Var. 1668 Var. 1668 0.7-0.9 Var. 1253 Var. 1669 Var. 1669 Var. 1669Var. 1669 Var. 1669 Var. 1669 Var. 1669 Var. 1669 0.1 Var. 1254 Var.1670 Var. 1670 Var. 1670 Var. 1670 Var. 1670 Var. 1670 Var. 1670 Var.1670 0.2 Var. 1255 Var. 1671 Var. 1671 Var. 1671 Var. 1671 Var. 1671Var. 1671 Var. 1671 Var. 1671 0.3 Var. 1256 Var. 1672 Var. 1672 Var.1672 Var. 1672 Var. 1672 Var. 1672 Var. 1672 Var. 1672 0.4 Var. 1257Var. 1673 Var. 1673 Var. 1673 Var. 1673 Var. 1673 Var. 1673 Var. 1673Var. 1673 0.5 Var. 1258 Var. 1674 Var. 1674 Var. 1674 Var. 1674 Var.1674 Var. 1674 Var. 1674 Var. 1674 0.6 Var. 1259 Var. 1675 Var. 1675Var. 1675 Var. 1675 Var. 1675 Var. 1675 Var. 1675 Var. 1675 0.7 Var.1260 Var. 1676 Var. 1676 Var. 1676 Var. 1676 Var. 1676 Var. 1676 Var.1676 Var. 1676 0.8 Var. 1261 Var. 1677 Var. 1677 Var. 1677 Var. 1677Var. 1677 Var. 1677 Var. 1677 Var. 1677 0.9 Var. 1262 Var. 1678 Var.1678 Var. 1678 Var. 1678 Var. 1678 Var. 1678 Var. 1678 Var. 1678 1 Var.1263 Var. 1679 Var. 1679 Var. 1679 Var. 1679 Var. 1679 Var. 1679 Var.1679 Var. 1679 1.1 Var. 1264 Var. 1680 Var. 1680 Var. 1680 Var. 1680Var. 1680 Var. 1680 Var. 1680 Var. 1680 1.2 Var. 1265 Var. 1681 Var.1681 Var. 1681 Var. 1681 Var. 1681 Var. 1681 Var. 1681 Var. 1681 1.3Var. 1266 Var. 1682 Var. 1682 Var. 1682 Var. 1682 Var. 1682 Var. 1682Var. 1682 Var. 1682 1.4 Var. 1267 Var. 1683 Var. 1683 Var. 1683 Var.1683 Var. 1683 Var. 1683 Var. 1683 Var. 1683 1.5 Var. 1268 Var. 1684Var. 1684 Var. 1684 Var. 1684 Var. 1684 Var. 1684 Var. 1684 Var. 16841.6 Var. 1269 Var. 1685 Var. 1685 Var. 1685 Var. 1685 Var. 1685 Var.1685 Var. 1685 Var. 1685 1.7 Var. 1270 Var. 1686 Var. 1686 Var. 1686Var. 1686 Var. 1686 Var. 1686 Var. 1686 Var. 1686 1.8 Var. 1271 Var.1687 Var. 1687 Var. 1687 Var. 1687 Var. 1687 Var. 1687 Var. 1687 Var.1687 1.9 Var. 1272 Var. 1688 Var. 1688 Var. 1688 Var. 1688 Var. 1688Var. 1688 Var. 1688 Var. 1688 2 Var. 1273 Var. 1689 Var. 1689 Var. 1689Var. 1689 Var. 1689 Var. 1689 Var. 1689 Var. 1689

TABLE 13 Exemplary embodiments of solution conditions useful for bindingserine proteases and/or serine protease zymogens to SiO₂. pH 5.0-7.05.0-6.5 5.0-6.0 5.0-5.5 4.6-5.6 4.7-5.5 4.8-5.4 4.9-5.3 5.0-5.2 Ionic0.1-2.0 Var. 1690 Var. 1742 Var. 1794 Var. 1846 Var. 1898 Var. 1950 Var.2002 Var. 2054 Var. 2106 Strength 0.1-1.9 Var. 1691 Var. 1743 Var. 1795Var. 1847 Var. 1899 Var. 1951 Var. 2003 Var. 2055 Var. 2107 (mS/cm)0.1-1.8 Var. 1692 Var. 1744 Var. 1796 Var. 1848 Var. 1900 Var. 1952 Var.2004 Var. 2056 Var. 2108 0.1-1.7 Var. 1693 Var. 1745 Var. 1797 Var. 1849Var. 1901 Var. 1953 Var. 2005 Var. 2057 Var. 2109 0.1-1.6 Var. 1694 Var.1746 Var. 1798 Var. 1850 Var. 1902 Var. 1954 Var. 2006 Var. 2058 Var.2110 0.1-1.5 Var. 1695 Var. 1747 Var. 1799 Var. 1851 Var. 1903 Var. 1955Var. 2007 Var. 2059 Var. 2111 0.1-1.4 Var. 1696 Var. 1748 Var. 1800 Var.1852 Var. 1904 Var. 1956 Var. 2008 Var. 2060 Var. 2112 0.1-1.3 Var. 1697Var. 1749 Var. 1801 Var. 1853 Var. 1905 Var. 1957 Var. 2009 Var. 2061Var. 2113 0.1-1.2 Var. 1698 Var. 1750 Var. 1802 Var. 1854 Var. 1906 Var.1958 Var. 2010 Var. 2062 Var. 2114 0.1-1.1 Var. 1699 Var. 1751 Var. 1803Var. 1855 Var. 1907 Var. 1959 Var. 2011 Var. 2063 Var. 2115 0.1-1.0 Var.1700 Var. 1752 Var. 1804 Var. 1856 Var. 1908 Var. 1960 Var. 2012 Var.2064 Var. 2116 0.1-0.9 Var. 1701 Var. 1753 Var. 1805 Var. 1857 Var. 1909Var. 1961 Var. 2013 Var. 2065 Var. 2117 0.1-0.8 Var. 1702 Var. 1754 Var.1806 Var. 1858 Var. 1910 Var. 1962 Var. 2014 Var. 2066 Var. 2118 0.2-2.0Var. 1703 Var. 1755 Var. 1807 Var. 1859 Var. 1911 Var. 1963 Var. 2015Var. 2067 Var. 2119 0.2-1.5 Var. 1704 Var. 1756 Var. 1808 Var. 1860 Var.1912 Var. 1964 Var. 2016 Var. 2068 Var. 2120 0.2-1.0 Var. 1705 Var. 1757Var. 1809 Var. 1861 Var. 1913 Var. 1965 Var. 2017 Var. 2069 Var. 21210.2-0.9 Var. 1706 Var. 1758 Var. 1810 Var. 1862 Var. 1914 Var. 1966 Var.2018 Var. 2070 Var. 2122 0.2-0.8 Var. 1707 Var. 1759 Var. 1811 Var. 1863Var. 1915 Var. 1967 Var. 2019 Var. 2071 Var. 2123 0.3-1.0 Var. 1708 Var.1760 Var. 1812 Var. 1864 Var. 1916 Var. 1968 Var. 2020 Var. 2072 Var.2124 0.3-0.9 Var. 1709 Var. 1761 Var. 1813 Var. 1865 Var. 1917 Var. 1969Var. 2021 Var. 2073 Var. 2125 0.3-0.8 Var. 1710 Var. 1762 Var. 1814 Var.1866 Var. 1918 Var. 1970 Var. 2022 Var. 2074 Var. 2126 0.4-1.0 Var. 1711Var. 1763 Var. 1815 Var. 1867 Var. 1919 Var. 1971 Var. 2023 Var. 2075Var. 2127 0.4-0.9 Var. 1712 Var. 1764 Var. 1816 Var. 1868 Var. 1920 Var.1972 Var. 2024 Var. 2076 Var. 2128 0.4-0.8 Var. 1713 Var. 1765 Var. 1817Var. 1869 Var. 1921 Var. 1973 Var. 2025 Var. 2077 Var. 2129 0.5-1.0 Var.1714 Var. 1766 Var. 1818 Var. 1870 Var. 1922 Var. 1974 Var. 2026 Var.2078 Var. 2130 0.5-0.9 Var. 1715 Var. 1767 Var. 1819 Var. 1871 Var. 1923Var. 1975 Var. 2027 Var. 2079 Var. 2131 0.5-0.8 Var. 1716 Var. 1768 Var.1820 Var. 1872 Var. 1924 Var. 1976 Var. 2028 Var. 2080 Var. 2132 0.6-1.0Var. 1717 Var. 1769 Var. 1821 Var. 1873 Var. 1925 Var. 1977 Var. 2029Var. 2081 Var. 2133 0.6-0.9 Var. 1718 Var. 1770 Var. 1822 Var. 1874 Var.1926 Var. 1978 Var. 2030 Var. 2082 Var. 2134 0.6-0.8 Var. 1719 Var. 1771Var. 1823 Var. 1875 Var. 1927 Var. 1979 Var. 2031 Var. 2083 Var. 21350.7-1.0 Var. 1720 Var. 1772 Var. 1824 Var. 1876 Var. 1928 Var. 1980 Var.2032 Var. 2084 Var. 2136 0.7-0.9 Var. 1721 Var. 1773 Var. 1825 Var. 1877Var. 1929 Var. 1981 Var. 2033 Var. 2085 Var. 2137 0.1 Var. 1722 Var.1774 Var. 1826 Var. 1878 Var. 1930 Var. 1982 Var. 2034 Var. 2086 Var.2138 0.2 Var. 1723 Var. 1775 Var. 1827 Var. 1879 Var. 1931 Var. 1983Var. 2035 Var. 2087 Var. 2139 0.3 Var. 1724 Var. 1776 Var. 1828 Var.1880 Var. 1932 Var. 1984 Var. 2036 Var. 2088 Var. 2140 0.4 Var. 1725Var. 1777 Var. 1829 Var. 1881 Var. 1933 Var. 1985 Var. 2037 Var. 2089Var. 2141 0.5 Var. 1726 Var. 1778 Var. 1830 Var. 1882 Var. 1934 Var.1986 Var. 2038 Var. 2090 Var. 2142 0.6 Var. 1727 Var. 1779 Var. 1831Var. 1883 Var. 1935 Var. 1987 Var. 2039 Var. 2091 Var. 2143 0.7 Var.1728 Var. 1780 Var. 1832 Var. 1884 Var. 1936 Var. 1988 Var. 2040 Var.2092 Var. 2144 0.8 Var. 1729 Var. 1781 Var. 1833 Var. 1885 Var. 1937Var. 1989 Var. 2041 Var. 2093 Var. 2145 0.9 Var. 1730 Var. 1782 Var.1834 Var. 1886 Var. 1938 Var. 1990 Var. 2042 Var. 2094 Var. 2146 1 Var.1731 Var. 1783 Var. 1835 Var. 1887 Var. 1939 Var. 1991 Var. 2043 Var.2095 Var. 2147 1.1 Var. 1732 Var. 1784 Var. 1836 Var. 1888 Var. 1940Var. 1992 Var. 2044 Var. 2096 Var. 2148 1.2 Var. 1733 Var. 1785 Var.1837 Var. 1889 Var. 1941 Var. 1993 Var. 2045 Var. 2097 Var. 2149 1.3Var. 1734 Var. 1786 Var. 1838 Var. 1890 Var. 1942 Var. 1994 Var. 2046Var. 2098 Var. 2150 1.4 Var. 1735 Var. 1787 Var. 1839 Var. 1891 Var.1943 Var. 1995 Var. 2047 Var. 2099 Var. 2151 1.5 Var. 1736 Var. 1788Var. 1840 Var. 1892 Var. 1944 Var. 1996 Var. 2048 Var. 2100 Var. 21521.6 Var. 1737 Var. 1789 Var. 1841 Var. 1893 Var. 1945 Var. 1997 Var.2049 Var. 2101 Var. 2153 1.7 Var. 1738 Var. 1790 Var. 1842 Var. 1894Var. 1946 Var. 1998 Var. 2050 Var. 2102 Var. 2154 1.8 Var. 1739 Var.1791 Var. 1843 Var. 1895 Var. 1947 Var. 1999 Var. 2051 Var. 2103 Var.2155 1.9 Var. 1740 Var. 1792 Var. 1844 Var. 1896 Var. 1948 Var. 2000Var. 2052 Var. 2104 Var. 2156 2 Var. 1741 Var. 1793 Var. 1845 Var. 1897Var. 1949 Var. 2001 Var. 2053 Var. 2105 Var. 2157

TABLE 14 Exemplary embodiments of solution conditions useful for bindingserine proteases and/or serine protease zymogens to SiO₂. pH NMT NMT NMTNMT NMT NMT NMT NMT 5.1 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 Ionic 0.1-2.0Var. 2158 Var. 2210 Var. 2262 Var. 2314 Var. 2366 Var. 2418 Var. 2470Var. 2522 Var. 2574 Strength 0.1-1.9 Var. 2159 Var. 2211 Var. 2263 Var.2315 Var. 2367 Var. 2419 Var. 2471 Var. 2523 Var. 2575 (mS/cm) 0.1-1.8Var. 2160 Var. 2212 Var. 2264 Var. 2316 Var. 2368 Var. 2420 Var. 2472Var. 2524 Var. 2576 0.1-1.7 Var. 2161 Var. 2213 Var. 2265 Var. 2317 Var.2369 Var. 2421 Var. 2473 Var. 2525 Var. 2577 0.1-1.6 Var. 2162 Var. 2214Var. 2266 Var. 2318 Var. 2370 Var. 2422 Var. 2474 Var. 2526 Var. 25780.1-1.5 Var. 2163 Var. 2215 Var. 2267 Var. 2319 Var. 2371 Var. 2423 Var.2475 Var. 2527 Var. 2579 0.1-1.4 Var. 2164 Var. 2216 Var. 2268 Var. 2320Var. 2372 Var. 2424 Var. 2476 Var. 2528 Var. 2580 0.1-1.3 Var. 2165 Var.2217 Var. 2269 Var. 2321 Var. 2373 Var. 2425 Var. 2477 Var. 2529 Var.2581 0.1-1.2 Var. 2166 Var. 2218 Var. 2270 Var. 2322 Var. 2374 Var. 2426Var. 2478 Var. 2530 Var. 2582 0.1-1.1 Var. 2167 Var. 2219 Var. 2271 Var.2323 Var. 2375 Var. 2427 Var. 2479 Var. 2531 Var. 2583 0.1-1.0 Var. 2168Var. 2220 Var. 2272 Var. 2324 Var. 2376 Var. 2428 Var. 2480 Var. 2532Var. 2584 0.1-0.9 Var. 2169 Var. 2221 Var. 2273 Var. 2325 Var. 2377 Var.2429 Var. 2481 Var. 2533 Var. 2585 0.1-0.8 Var. 2170 Var. 2222 Var. 2274Var. 2326 Var. 2378 Var. 2430 Var. 2482 Var. 2534 Var. 2586 0.2-2.0 Var.2171 Var. 2223 Var. 2275 Var. 2327 Var. 2379 Var. 2431 Var. 2483 Var.2535 Var. 2587 0.2-1.5 Var. 2172 Var. 2224 Var. 2276 Var. 2328 Var. 2380Var. 2432 Var. 2484 Var. 2536 Var. 2588 0.2-1.0 Var. 2173 Var. 2225 Var.2277 Var. 2329 Var. 2381 Var. 2433 Var. 2485 Var. 2537 Var. 2589 0.2-0.9Var. 2174 Var. 2226 Var. 2278 Var. 2330 Var. 2382 Var. 2434 Var. 2486Var. 2538 Var. 2590 0.2-0.8 Var. 2175 Var. 2227 Var. 2279 Var. 2331 Var.2383 Var. 2435 Var. 2487 Var. 2539 Var. 2591 0.3-1.0 Var. 2176 Var. 2228Var. 2280 Var. 2332 Var. 2384 Var. 2436 Var. 2488 Var. 2540 Var. 25920.3-0.9 Var. 2177 Var. 2229 Var. 2281 Var. 2333 Var. 2385 Var. 2437 Var.2489 Var. 2541 Var. 2593 0.3-0.8 Var. 2178 Var. 2230 Var. 2282 Var. 2334Var. 2386 Var. 2438 Var. 2490 Var. 2542 Var. 2594 0.4-1.0 Var. 2179 Var.2231 Var. 2283 Var. 2335 Var. 2387 Var. 2439 Var. 2491 Var. 2543 Var.2595 0.4-0.9 Var. 2180 Var. 2232 Var. 2284 Var. 2336 Var. 2388 Var. 2440Var. 2492 Var. 2544 Var. 2596 0.4-0.8 Var. 2181 Var. 2233 Var. 2285 Var.2337 Var. 2389 Var. 2441 Var. 2493 Var. 2545 Var. 2597 0.5-1.0 Var. 2182Var. 2234 Var. 2286 Var. 2338 Var. 2390 Var. 2442 Var. 2494 Var. 2546Var. 2598 0.5-0.9 Var. 2183 Var. 2235 Var. 2287 Var. 2339 Var. 2391 Var.2443 Var. 2495 Var. 2547 Var. 2599 0.5-0.8 Var. 2184 Var. 2236 Var. 2288Var. 2340 Var. 2392 Var. 2444 Var. 2496 Var. 2548 Var. 2600 0.6-1.0 Var.2185 Var. 2237 Var. 2289 Var. 2341 Var. 2393 Var. 2445 Var. 2497 Var.2549 Var. 2601 0.6-0.9 Var. 2186 Var. 2238 Var. 2290 Var. 2342 Var. 2394Var. 2446 Var. 2498 Var. 2550 Var. 2602 0.6-0.8 Var. 2187 Var. 2239 Var.2291 Var. 2343 Var. 2395 Var. 2447 Var. 2499 Var. 2551 Var. 2603 0.7-1.0Var. 2188 Var. 2240 Var. 2292 Var. 2344 Var. 2396 Var. 2448 Var. 2500Var. 2552 Var. 2604 0.7-0.9 Var. 2189 Var. 2241 Var. 2293 Var. 2345 Var.2397 Var. 2449 Var. 2501 Var. 2553 Var. 2605 0.1 Var. 2190 Var. 2242Var. 2294 Var. 2346 Var. 2398 Var. 2450 Var. 2502 Var. 2554 Var. 26060.2 Var. 2191 Var. 2243 Var. 2295 Var. 2347 Var. 2399 Var. 2451 Var.2503 Var. 2555 Var. 2607 0.3 Var. 2192 Var. 2244 Var. 2296 Var. 2348Var. 2400 Var. 2452 Var. 2504 Var. 2556 Var. 2608 0.4 Var. 2193 Var.2245 Var. 2297 Var. 2349 Var. 2401 Var. 2453 Var. 2505 Var. 2557 Var.2609 0.5 Var. 2194 Var. 2246 Var. 2298 Var. 2350 Var. 2402 Var. 2454Var. 2506 Var. 2558 Var. 2610 0.6 Var. 2195 Var. 2247 Var. 2299 Var.2351 Var. 2403 Var. 2455 Var. 2507 Var. 2559 Var. 2611 0.7 Var. 2196Var. 2248 Var. 2300 Var. 2352 Var. 2404 Var. 2456 Var. 2508 Var. 2560Var. 2612 0.8 Var. 2197 Var. 2249 Var. 2301 Var. 2353 Var. 2405 Var.2457 Var. 2509 Var. 2561 Var. 2613 0.9 Var. 2198 Var. 2250 Var. 2302Var. 2354 Var. 2406 Var. 2458 Var. 2510 Var. 2562 Var. 2614 1 Var. 2199Var. 2251 Var. 2303 Var. 2355 Var. 2407 Var. 2459 Var. 2511 Var. 2563Var. 2615 1.1 Var. 2200 Var. 2252 Var. 2304 Var. 2356 Var. 2408 Var.2460 Var. 2512 Var. 2564 Var. 2616 1.2 Var. 2201 Var. 2253 Var. 2305Var. 2357 Var. 2409 Var. 2461 Var. 2513 Var. 2565 Var. 2617 1.3 Var.2202 Var. 2254 Var. 2306 Var. 2358 Var. 2410 Var. 2462 Var. 2514 Var.2566 Var. 2618 1.4 Var. 2203 Var. 2255 Var. 2307 Var. 2359 Var. 2411Var. 2463 Var. 2515 Var. 2567 Var. 2619 1.5 Var. 2204 Var. 2256 Var.2308 Var. 2360 Var. 2412 Var. 2464 Var. 2516 Var. 2568 Var. 2620 1.6Var. 2205 Var. 2257 Var. 2309 Var. 2361 Var. 2413 Var. 2465 Var. 2517Var. 2569 Var. 2621 1.7 Var. 2206 Var. 2258 Var. 2310 Var. 2362 Var.2414 Var. 2466 Var. 2518 Var. 2570 Var. 2622 1.8 Var. 2207 Var. 2259Var. 2311 Var. 2363 Var. 2415 Var. 2467 Var. 2519 Var. 2571 Var. 26231.9 Var. 2208 Var. 2260 Var. 2312 Var. 2364 Var. 2416 Var. 2468 Var.2520 Var. 2572 Var. 2624 2 Var. 2209 Var. 2261 Var. 2313 Var. 2365 Var.2417 Var. 2469 Var. 2521 Var. 2573 Var. 2625 NMT = No More Than

TABLE 15 Exemplary embodiments of solution conditions useful for bindingserine proteases and/or serine protease zymogens to SiO₂. pH NMT 5.6 NMT5.8 NMT 6.0 NMT 6.2 NMT 6.4 NMT 6.6 NMT 6.8 NMT 7.0 Ionic 0.1-2.0 Var.2626 Var. 2678 Var. 2730 Var. 2782 Var. 2834 Var. 2886 Var. 2938 Var.2990 Strength 0.1-1.9 Var. 2627 Var. 2679 Var. 2731 Var. 2783 Var. 2835Var. 2887 Var. 2939 Var. 2991 (mS/cm) 0.1-1.8 Var. 2628 Var. 2680 Var.2732 Var. 2784 Var. 2836 Var. 2888 Var. 2940 Var. 2992 0.1-1.7 Var. 2629Var. 2681 Var. 2733 Var. 2785 Var. 2837 Var. 2889 Var. 2941 Var. 29930.1-1.6 Var. 2630 Var. 2682 Var. 2734 Var. 2786 Var. 2838 Var. 2890 Var.2942 Var. 2994 0.1-1.5 Var. 2631 Var. 2683 Var. 2735 Var. 2787 Var. 2839Var. 2891 Var. 2943 Var. 2995 0.1-1.4 Var. 2632 Var. 2684 Var. 2736 Var.2788 Var. 2840 Var. 2892 Var. 2944 Var. 2996 0.1-1.3 Var. 2633 Var. 2685Var. 2737 Var. 2789 Var. 2841 Var. 2893 Var. 2945 Var. 2997 0.1-1.2 Var.2634 Var. 2686 Var. 2738 Var. 2790 Var. 2842 Var. 2894 Var. 2946 Var.2998 0.1-1.1 Var. 2635 Var. 2687 Var. 2739 Var. 2791 Var. 2843 Var. 2895Var. 2947 Var. 2999 0.1-1.0 Var. 2636 Var. 2688 Var. 2740 Var. 2792 Var.2844 Var. 2896 Var. 2948 Var. 3000 0.1-0.9 Var. 2637 Var. 2689 Var. 2741Var. 2793 Var. 2845 Var. 2897 Var. 2949 Var. 3001 0.1-0.8 Var. 2638 Var.2690 Var. 2742 Var. 2794 Var. 2846 Var. 2898 Var. 2950 Var. 3002 0.2-2.0Var. 2639 Var. 2691 Var. 2743 Var. 2795 Var. 2847 Var. 2899 Var. 2951Var. 3003 0.2-1.5 Var. 2640 Var. 2692 Var. 2744 Var. 2796 Var. 2848 Var.2900 Var. 2952 Var. 3004 0.2-1.0 Var. 2641 Var. 2693 Var. 2745 Var. 2797Var. 2849 Var. 2901 Var. 2953 Var. 3005 0.2-0.9 Var. 2642 Var. 2694 Var.2746 Var. 2798 Var. 2850 Var. 2902 Var. 2954 Var. 3006 0.2-0.8 Var. 2643Var. 2695 Var. 2747 Var. 2799 Var. 2851 Var. 2903 Var. 2955 Var. 30070.3-1.0 Var. 2644 Var. 2696 Var. 2748 Var. 2800 Var. 2852 Var. 2904 Var.2956 Var. 3008 0.3-0.9 Var. 2645 Var. 2697 Var. 2749 Var. 2801 Var. 2853Var. 2905 Var. 2957 Var. 3009 0.3-0.8 Var. 2646 Var. 2698 Var. 2750 Var.2802 Var. 2854 Var. 2906 Var. 2958 Var. 3010 0.4-1.0 Var. 2647 Var. 2699Var. 2751 Var. 2803 Var. 2855 Var. 2907 Var. 2959 Var. 3011 0.4-0.9 Var.2648 Var. 2700 Var. 2752 Var. 2804 Var. 2856 Var. 2908 Var. 2960 Var.3012 0.4-0.8 Var. 2649 Var. 2701 Var. 2753 Var. 2805 Var. 2857 Var. 2909Var. 2961 Var. 3013 0.5-1.0 Var. 2650 Var. 2702 Var. 2754 Var. 2806 Var.2858 Var. 2910 Var. 2962 Var. 3014 0.5-0.9 Var. 2651 Var. 2703 Var. 2755Var. 2807 Var. 2859 Var. 2911 Var. 2963 Var. 3015 0.5-0.8 Var. 2652 Var.2704 Var. 2756 Var. 2808 Var. 2860 Var. 2912 Var. 2964 Var. 3016 0.6-1.0Var. 2653 Var. 2705 Var. 2757 Var. 2809 Var. 2861 Var. 2913 Var. 2965Var. 3017 0.6-0.9 Var. 2654 Var. 2706 Var. 2758 Var. 2810 Var. 2862 Var.2914 Var. 2966 Var. 3018 0.6-0.8 Var. 2655 Var. 2707 Var. 2759 Var. 2811Var. 2863 Var. 2915 Var. 2967 Var. 3019 0.7-1.0 Var. 2656 Var. 2708 Var.2760 Var. 2812 Var. 2864 Var. 2916 Var. 2968 Var. 3020 0.7-0.9 Var. 2657Var. 2709 Var. 2761 Var. 2813 Var. 2865 Var. 2917 Var. 2969 Var. 30210.1 Var. 2658 Var. 2710 Var. 2762 Var. 2814 Var. 2866 Var. 2918 Var.2970 Var. 3022 0.2 Var. 2659 Var. 2711 Var. 2763 Var. 2815 Var. 2867Var. 2919 Var. 2971 Var. 3023 0.3 Var. 2660 Var. 2712 Var. 2764 Var.2816 Var. 2868 Var. 2920 Var. 2972 Var. 3024 0.4 Var. 2661 Var. 2713Var. 2765 Var. 2817 Var. 2869 Var. 2921 Var. 2973 Var. 3025 0.5 Var.2662 Var. 2714 Var. 2766 Var. 2818 Var. 2870 Var. 2922 Var. 2974 Var.3026 0.6 Var. 2663 Var. 2715 Var. 2767 Var. 2819 Var. 2871 Var. 2923Var. 2975 Var. 3027 0.7 Var. 2664 Var. 2716 Var. 2768 Var. 2820 Var.2872 Var. 2924 Var. 2976 Var. 3028 0.8 Var. 2665 Var. 2717 Var. 2769Var. 2821 Var. 2873 Var. 2925 Var. 2977 Var. 3029 0.9 Var. 2666 Var.2718 Var. 2770 Var. 2822 Var. 2874 Var. 2926 Var. 2978 Var. 3030 1 Var.2667 Var. 2719 Var. 2771 Var. 2823 Var. 2875 Var. 2927 Var. 2979 Var.3031 1.1 Var. 2668 Var. 2720 Var. 2772 Var. 2824 Var. 2876 Var. 2928Var. 2980 Var. 3032 1.2 Var. 2669 Var. 2721 Var. 2773 Var. 2825 Var.2877 Var. 2929 Var. 2981 Var. 3033 1.3 Var. 2670 Var. 2722 Var. 2774Var. 2826 Var. 2878 Var. 2930 Var. 2982 Var. 3034 1.4 Var. 2671 Var.2723 Var. 2775 Var. 2827 Var. 2879 Var. 2931 Var. 2983 Var. 3035 1.5Var. 2672 Var. 2724 Var. 2776 Var. 2828 Var. 2880 Var. 2932 Var. 2984Var. 3036 1.6 Var. 2673 Var. 2725 Var. 2777 Var. 2829 Var. 2881 Var.2933 Var. 2985 Var. 3037 1.7 Var. 2674 Var. 2726 Var. 2778 Var. 2830Var. 2882 Var. 2934 Var. 2986 Var. 3038 1.8 Var. 2675 Var. 2727 Var.2779 Var. 2831 Var. 2883 Var. 2935 Var. 2987 Var. 3039 1.9 Var. 2676Var. 2728 Var. 2780 Var. 2832 Var. 2884 Var. 2936 Var. 2988 Var. 3040 2Var. 2677 Var. 2729 Var. 2781 Var. 2833 Var. 2885 Var. 2937 Var. 2989Var. 3041 NMT = No More Than

A. Modified Alcohol Precipitation/Ion Exchange ChromatographyFractionation Methods

In one aspect, the present invention provides improved methods for themanufacture of IgG compositions suitable for use in IVIG therapy.Generally, these methods provide IgG preparations having higher yieldsand comparable if not higher purity than current methods employed forthe production of commercial IVIG products.

In one specific aspect, the present invention provides a method forpreparing a composition of concentrated IgG from plasma, e.g., 10% IVIG,the method comprising performing at least one alcohol precipitation stepand at least one ion exchange chromatography step. In particular,several steps in the improved upstream process are different from priorprocesses, e.g., the use of 25% ethanol at lower temperatures, ethanoladdition by spraying, pH adjustment by spraying, and the use of finelydivided silica particles.

In a certain embodiment, the method comprises the steps of (a)precipitating a cryo-poor plasma fraction, in a first precipitationstep, with between about 6% and about 10% alcohol at a pH of betweenabout 6.7 and about 7.3 to obtain a supernatant enriched in IgG, (b)precipitating IgG from the supernatant with between about 20% and about30% alcohol at a lower temperature and at a pH of between about 6.7 andabout 7.3 to form a first precipitate, (c) re-suspending the firstprecipitate formed in step (b) to form a suspension, (d) treating thesuspension formed in step (c) with a detergent, (e) precipitating IgGfrom the suspension with between about 20% and about 30% alcohol at a pHof between about 6.7 and about 7.3 to form a second precipitate, (f)re-suspending the second precipitate formed in step (e) to form asuspension, (g) treating the suspension formed in step (f) with asolvent and/or detergent, and (h) performing at least one ion exchangechromatography fractionation thereby preparing a composition ofconcentrated IgG. In one embodiment, the method further comprisestreating the suspension formed in step (c) with finely divided silicadioxide (SiO₂) and filtering the solution prior to step (d).

In one embodiment, a method for preparing a concentrated IgG compositionfrom plasma is provided, the method comprising the steps of (a)adjusting the pH of a cryo-poor plasma fraction to about 7.0, (b)adjusting the ethanol concentration of the cryo-poor plasma fraction ofstep (a) to at or about 25% (v/v) at a temperature between about −5° C.and about −9° C., thereby forming a mixture, wherein the ethanolconcentration may be adjusted by spraying, (c) separating liquid andprecipitate from the mixture of step (b), (d) re-suspending theprecipitate of step (c) with a buffer containing phosphate and acetate,wherein the pH of the buffer is adjusted with between about 400 andabout 700 ml of glacial acetic acid per 1000 L of buffer, therebyforming a suspension, (e) mixing finely divided silicon dioxide (SiO2)with the suspension from step (d) for at least about 30 minutes, (f)filtering the suspension with a filter press, thereby forming afiltrate, (g) washing the filter press with at least 3 filter press deadvolumes of a buffer containing phosphate and acetate, wherein the pH ofthe buffer is adjusted with about 150 ml of glacial acetic acid per 1000L of buffer, thereby forming a wash solution, (h) combining the filtrateof step (f) with the wash solution of step (g), thereby forming asolution, and treating the solution with a detergent, (i) adjusting thepH of the solution of step (h) to about 7.0 and adding ethanol to afinal concentration of at or about 25%, thereby forming a precipitate,wherein the ethanol concentration and/or pH may be adjusted by spraying(j) separating liquid and precipitate from the mixture of step (i), (k)dissolving the precipitate in an aqueous solution comprising a solventor detergent and maintaining the solution for at least 60 minutes, (l)passing the solution after step (k) through a cation exchangechromatography column and eluting proteins absorbed on the column in aneluate, (m) passing the eluate from step (l) through an anion exchangechromatography column to generate an effluent (i.e., flow-through), (n)passing the effluent from step (m) through a nanofilter to generate ananofiltrate, (o) passing the nanofiltrate from step (n) through anultrafiltration membrane to generate an ultrafiltrate, and (p)diafiltrating the ultrafiltrate from step (o) against a diafiltrationbuffer to generate a diafiltrate having a protein concentration betweenabout 8% (w/v) and about 22% (w/v), thereby obtaining a composition ofconcentrated IgG. In one embodiment, the temperature of step (b) is ator about −7° C. In one specific embodiment, the suspension buffer instep (d) is adjusted with about 600 mL glacial acetic acid.

In certain embodiments, the diafiltrate will have a proteinconcentration between about 8% and about 12%, for example, about 8%, orabout 9%, 10%, 11%, or 12%. In a preferred embodiment, the diafiltratewill have a protein concentration of at or about 10%. In anotherpreferred embodiment, the diafiltrate will have a protein concentrationof at or about 11%. In yet another preferred embodiment, the diafiltratewill have a protein concentration of at or about 12%. In otherembodiments, the diafiltrate will have a protein concentration betweenabout 13% and about 17%, for example, about 13%, or about 14%, 15%, 16%,or 17%. In yet other embodiments, the diafiltrate will have a proteinconcentration between about 18% and about 22%, for example, about 18%,or about 19%, 20%, 21%, or 22%. In a preferred embodiment, thediafiltrate will have a protein concentration of at or about 20%. Inanother preferred embodiment, the diafiltrate will have a proteinconcentration of at or about 21%. In yet another preferred embodiment,the diafiltrate will have a protein concentration of at or about 22%.

In certain embodiments of the present invention, the methods providedherein may comprise improvements in two or more of the fractionationprocess steps described above. For example, embodiments may includeimprovements in the first precipitation step, the Modified FractionII+III precipitation step, the Modified Fraction II+III dissolutionstep, and/or the Modified Fraction II+III suspension filtration step.

In one embodiment, the improvement made in the first precipitation stepis the addition of alcohol by spraying. In another embodiment, theimprovement made in the first precipitation step is the addition of a pHmodifying agent by spraying. In yet embodiment, the improvement made inthe first precipitation step is the adjustment of the pH of the solutionafter addition of the alcohol. In a related embodiment, the improvementmade in the first precipitation step is the maintenance of the pH duringthe addition of the alcohol. In another related embodiment, theimprovement made in the first precipitation step is the maintenance ofthe pH during the precipitation incubation time by continuouslyadjusting the pH of the solution. In certain embodiments, the firstprecipitation step may be improved by implementing more than one ofthese improvements. Further improvements that may be realized in thisstep will be evident from the section provided below discussing thefirst precipitation step—Modified Fractionation I. By implementing oneor more of the improvements described above, a reduced amount of IgG islost in the precipitate fraction of the first precipitation step and/ora reduced fraction of IgG is irreversibly denatured during theprecipitation step.

In one embodiment, the improvement made in the Modified Fraction II+IIIprecipitation step is the addition of alcohol by spraying. In anotherembodiment, the improvement made in the Modified Fraction II+IIIprecipitation step is the addition of a pH modifying agent by spraying.In yet embodiment, the improvement made in the Modified Fraction II+IIIprecipitation step is the adjustment of the pH of the solution afteraddition of the alcohol. In a related embodiment, the improvement madein the Modified Fraction II+III precipitation step is the maintenance ofthe pH during the addition of the alcohol. In another relatedembodiment, the improvement made in the Modified Fraction II+IIIprecipitation step is the maintenance of the pH during the precipitationincubation time by continuously adjusting the pH of the solution. Inanother aspect, the Modified Fraction II+III precipitation step isimproved by increasing the concentration of alcohol to at or about 25%.In yet another embodiment, the Modified Fraction II+III precipitationstep is improved by lowering the incubation temperature to between about−7° C. and −9° C. In certain embodiments, the Modified Fraction II+IIIprecipitation step may be improved by implementing more than one ofthese improvements. Further improvements that may be realized in thisstep will be evident from the section provided below discussing thesecond precipitation step—Modified Fractionation II+III. By implementingone or more of the improvements described above, a reduced amount of IgGis lost in the supernatant fraction of the Modified Fraction II+IIIprecipitation step and/or a reduced fraction of IgG is irreversiblydenatured during the precipitation step.

In one embodiment, the improvement made in the Modified Fraction II+IIIdissolution step is achieved by increasing the glacial acetic acidcontent of the dissolution buffer to about 0.06%. In another embodiment,the improvement made in the Modified Fraction II+III dissolution step isachieved by maintaining the pH of the solution during the dissolutionincubation time by continuously adjusting the pH of the solution. Inanother embodiment, the improvement made in the Modified Fraction II+IIIdissolution step is achieved by mixing finely divided silicon dioxide(SiO₂) with the Fraction II+III suspension prior to filtration. Incertain embodiments, the Modified Fraction II+III dissolution step maybe improved by implementing more than one of these improvements. Furtherimprovements that may be realized in this step will be evident from thesection provided below discussing the Modified Fraction II+IIIdissolution step—Extraction of the Modified Fraction II+III Precipitate.By implementing one or more of the improvements described above, anincreased amount of IgG is recovered in the Fraction II+III suspensionand/or the amount of impurities is reduced in the Fraction II+IIIsuspension.

An exemplary improvement made in the Modified Fraction II+III suspensionfiltration step is realized by post-washing the filter with at leastabout 3.6 dead volumes of dissolution buffer containing at or about 150mL glacial acetic acid per 1000 L. Further improvements that may berealized in this step will be evident from the section provided belowdiscussing the Modified Fraction II+III suspension filtrationstep—Pretreatment and Filtration of the Modified Fraction II+IIISuspension. By implementing one or more of the improvements describedabove, a reduced amount of IgG is lost during the Modified FractionII+III suspension filtration step.

In one embodiment, the method may comprise an improvement in the firstprecipitation step and the Modified Fraction II+III precipitation step.

In another embodiment, the method may comprise an improvement in thefirst precipitation step and the Modified Fraction II+III dissolutionstep.

In another embodiment, the method may comprise an improvement in thefirst precipitation step and the Modified Fraction II+III suspensionfiltration step.

In another embodiment, the method may comprise an improvement in theModified Fraction II+III precipitation step and the Modified FractionII+III dissolution step.

In another embodiment, the method may comprise an improvement in theModified Fraction II+III precipitation step and the Modified FractionII+III suspension filtration step.

In another embodiment, the method may comprise an improvement in theModified Fraction II+III dissolution step and the Modified FractionII+III suspension filtration step.

In another embodiment, the method may comprise an improvement in thefirst precipitation step, the Modified Fraction II+III precipitationstep, and the Modified Fraction II+III dissolution step.

In another embodiment, the method may comprise an improvement in thefirst precipitation step, the Modified Fraction II+III precipitationstep, and the Modified Fraction II+III suspension filtration step.

In another embodiment, the method may comprise an improvement in thefirst precipitation step, the Modified Fraction II+III dissolution step,and the Modified Fraction II+III suspension filtration step.

In another embodiment, the method may comprise an improvement in theModified Fraction II+III precipitation step, the Modified FractionII+III dissolution step, and the Modified Fraction II+III suspensionfiltration step.

In another embodiment, the method may comprise an improvement in all ofthe first precipitation step, the Modified Fraction II+III precipitationstep, the Modified Fraction II+III dissolution step, and the ModifiedFraction II+III suspension filtration step.

In certain embodiments, one process improvement in the IgG purificationmethods provided herein comprises the spray addition of one or moresolutions that would otherwise be introduced into a plasma fraction byfluent addition. For example, in certain embodiments the processimprovement comprises the addition of alcohol (e.g., ethanol) into aplasma fraction for the purposes of precipitation of one or more proteinspecies by spraying. In other embodiments, solutions that may be addedto a plasma fraction by spraying include, without limitation, a pHmodifying solution, a solvent solution, a detergent solution, a dilutionbuffer, a conductivity modifying solution, and the like. In a preferredembodiment, one or more alcohol precipitation steps is performed by theaddition of alcohol to a plasma fraction by spraying. In a secondpreferred embodiment, one or more pH adjustment steps is performed bythe addition of a pH modifying solution to a plasma fraction byspraying.

In certain embodiments, another process improvement, which may becombined with any other process improvement, comprises the adjustment ofthe pH of a plasma fraction being precipitated after and/or concomitantwith the addition of the precipitating agent (e.g., alcohol orpolyethelene glycol). In some embodiments, a process improvement isprovided in which the pH of a plasma fraction being activelyprecipitated is maintained throughout the entire precipitationincubation or hold step by continuous monitoring and adjustment of thepH. In preferred embodiments the adjustment of the pH is performed bythe spray addition of a pH modifying solution.

In other embodiments, another process improvement, which may be combinedwith any other process improvement, comprises the use of a finelydivided silica treatment step to remove impurities.

1. Preparation of Cryo-Poor Plasma

The starting material used for the preparation of concentrated IgGcompositions generally consists of either recovered plasma (i.e., plasmathat has been separated from whole blood ex vivo) or source plasma(i.e., plasma collected via plasmapheresis). The purification processtypically starts with thawing previously frozen pooled plasma, which hasalready been assayed for safety and quality considerations. Thawing istypically carried out at a temperature no higher than 6° C. Aftercomplete thawing of the frozen plasma at low temperature, centrifugationis performed in the cold (e.g., ≦6° C.) to separate solidcryo-precipitates from the liquid supernatant. Alternatively, theseparation step can be performed by filtration rather thancentrifugation. The liquid supernatant (also referred to as “cryo-poorplasma,” after cold-insoluble proteins removed by centrifugation fromfresh thawed plasma) is then processed in the next step. Variousadditional steps can be taken at this juncture for the isolation offactor eight inhibitor bypass activity (FEIBA), Factor IX-complex,Factor VII-concentrate, or Antithrombin III-complex.

2. First Precipitation Event—Modified Fractionation I

In this step, cryo-poor plasma is typically cooled to about 0±1° C. andthe pH is adjusted to between about 7.0 and about 7.5, preferablybetween about 7.1 and about 7.3, most preferably about 7.2. In oneembodiment, the pH of the cryo-poor plasma is adjusted to a pH of at orabout 7.2. Pre-cooled ethanol is then added while the plasma is stirredto a target concentration of ethanol at or about 8% v/v. At the sametime the temperature is further lowered to between about −4 and about 0°C. In a preferred embodiment, the temperature is lowered to at or about−2° C., to precipitate contaminants such as α₂-macroglobulin, β_(1A)-and β_(1C)-globulin, fibrinogen, and Factor VIII. Typically, theprecipitation event will include a hold time of at least about 1 hour,although shorter or longer hold times may also be employed.Subsequently, the supernatant (Supernatant I), ideally containing theentirety of the IgG content present in the cryo-poor plasma, is thencollected by centrifugation, filtration, or another suitable method.

As compared to conventional methods employed as a first fractionationstep for cryo-poor plasma (Cohn et al., supra; Oncley et al., supra),the present invention provides, in several embodiments, methods thatresult in improved IgG yields in the Supernatant I fraction. In oneembodiment, the improved IgG yield is achieved by adding the alcohol byspraying. In another embodiment, the improved IgG yield is achieved byadding a pH modifying agent by spraying. In yet another embodiment, theimproved IgG yield is achieved by adjusting the pH of the solution afteraddition of the alcohol. In a related embodiment, the improved IgG yieldis achieved by adjusting the pH of the solution during the addition ofthe alcohol.

In one specific aspect, the improvement relates to a method in which areduced amount of IgG is lost in the precipitate fraction of the firstprecipitation step. For example, in certain embodiments, a reducedamount of IgG is lost in the precipitate fraction of the firstprecipitation step as compared to the amount of IgG lost in the firstprecipitation step of the Cohn method 6 protocol.

In certain embodiments, the process improvement is realized by adjustingthe pH of the solution to between about 7.0 and about 7.5 after theaddition of the precipitating alcohol. In other embodiments, the pH ofthe solution is adjusted to between about 7.1 and about 7.3 afteraddition of the precipitating alcohol. In yet other embodiments, the pHof the solution is adjusted to about 7.0 or about 7.1, 7.2, 7.3, 7.4, or7.5 after addition of the precipitating alcohol. In a particularembodiment, the pH of the solution is adjusted to about 7.2 afteraddition of the precipitating alcohol. As such, in certain embodiments,a reduced amount of IgG is lost in the precipitate fraction of the firstprecipitation step as compared to an analogous precipitation step inwhich the pH of the solution is adjusted prior to but not after additionof the precipitating alcohol. In one embodiment, the pH is maintained atthe desired pH during the precipitation hold or incubation time bycontinuously adjusting the pH of the solution. In one embodiment, thealcohol is ethanol.

In other certain embodiments, the process improvement is realized byadding the precipitating alcohol and/or the solution used to adjust thepH by spraying, rather than by fluent addition. As such, in certainembodiments, a reduced amount of IgG is lost in the precipitate fractionof the first precipitation step as compared to an analogousprecipitation step in which the alcohol and/or solution used to adjustthe pH is introduced by fluent addition. In one embodiment, the alcoholis ethanol.

In yet other certain embodiments, the improvement is realized byadjusting the pH of the solution to between about 7.0 and about 7.5. Ina preferred embodiment, the pH of the solution is adjusted to betweenabout 7.1 and about 7.3. In other embodiments, the pH of the solution isadjusted to at or about 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 after theaddition of the precipitating alcohol and by adding the precipitatingalcohol and/or the solution used to adjust the pH by spraying, ratherthan by fluent addition. In a particular embodiment, the pH of thesolution is adjusted to at or about 7.2 after addition of theprecipitating alcohol and by adding the precipitating alcohol and/or thesolution used to adjust the pH by spraying, rather than by fluentaddition. In one embodiment, the alcohol is ethanol.

3. Second Precipitation Event—Modified Fractionation II+III

To further enrich the IgG content and purity of the fractionation,Supernatant I is subjected to a second precipitation step, which is amodified Cohn-Oncley Fraction II+III fractionation. Generally, the pH ofthe solution is adjusted to a pH of between about 6.6 and about 6.8. Ina preferred embodiment, the pH of the solution is adjusted to at orabout 6.7. Alcohol, preferably ethanol, is then added to the solutionwhile being stirred to a final concentration of between about 20% andabout 25% (v/v) to precipitate the IgG in the fraction. In a preferredembodiment, alcohol is added to a final concentration of at or about 25%(v/v) to precipitate the IgG in the fraction. Generally, contaminantssuch as α₁-lipoprotein, α₁-antitrypsin, Gc-globulins,α_(1X)-glycoprotin, haptoglobulin, ceruloplasmin, transferrin,hemopexin, a fraction of the Christmas factor, thyroxin bindingglobulin, cholinesterase, hypertensinogen, and albumin will not beprecipitated by these conditions.

Prior to or concomitant with alcohol addition, the solution is furthercooled to between about −7° C. and about −9° C. In a preferredembodiment, the solution is cooled to a temperature at or about −7° C.After completion of the alcohol addition, the pH of the solution isimmediately adjusted to between about 6.8 and about 7.0. In a preferredembodiment, the pH of the solution is adjusted to at or about 6.9.Typically, the precipitation event will include a hold time of at leastabout 10 hours, although shorter or longer hold times may also beemployed. Subsequently, the precipitate (Modified Fraction II+III),which ideally contains at least about 85%, preferably at least about90%, more preferably at least about 95%, of the IgG content present inthe cryo-poor plasma, is separated from the supernatant bycentrifugation, filtration, or another suitable method and collected. Ascompared to conventional methods employed as a second fractionation stepfor cryo-poor plasma (Cohn et al., supra; Oncley et al., supra), thepresent invention provides, in several embodiments, methods that resultin improved IgG yields in the Modified Fraction II+III precipitate. In arelated embodiment, the present invention provides methods that resultin a reduced loss of IgG in the Modified II+III supernatant.

As compared to conventional methods employed as a second fractionationstep for cryo-poor plasma (Cohn et al., supra; Oncley et al., supra),the present invention provides, in several embodiments, methods thatresult in improved IgG yields in the Modified Fraction II+IIIprecipitate. In one embodiment, the improvement is realized by theaddition of alcohol by spraying. In another embodiment, the improvementis realized by the addition of a pH modifying agent by spraying. Inanother embodiment, the improvement is realized by adjusting the pH ofthe solution after addition of the alcohol. In a related embodiment, theimprovement is realized by adjusting the pH of the solution duringaddition of the alcohol. In another embodiment, the improvement isrealized by increasing the concentration of alcohol (e.g., ethanol) toabout 25% (v/v). In another embodiment, the improvement is realized bylowering the temperature of the precipitation step to between about −7°C. and −9° C. In a preferred embodiment, the improvement is realized byincreasing the concentration of alcohol (e.g., ethanol) to about 25%(v/v) and lowing the temperature to between about −7° C. and −9° C. Incomparison, both Cohn et al. and Oncley et al. perform precipitation at−5° C. and Oncley et al. use 20% alcohol, in order to reduce the levelof contaminants in the precipitate. Advantageously, the methods providedherein allow for maximal IgG yield without high levels of contaminationin the final product.

It has been discovered that when the pH of the solution is adjusted to apH of about 6.9 prior to addition of the precipitating alcohol, the pHof the solution shift from 6.9 to between about 7.4 and about 7.7, duein part to protein precipitation (see, FIG. 8). As the pH of thesolution shifts away from 6.9, precipitation of IgG becomes lessfavorable and the precipitation of certain contaminants becomes morefavorable. Advantageously, the inventors have found that by adjustingthe pH of the solution after addition of the precipitating alcohol, thata higher percentage of IgG is recovered in the Fraction II+IIIprecipitate.

Accordingly, in one aspect, the improvement relates to a method in whicha reduced amount of IgG is lost in the supernatant fraction of themodified Fraction II+III precipitation step. In other words, anincreased percentage of the starting IgG is present in the FractionII+III precipitate. In certain embodiments, the process improvement isrealized by adjusting the pH of the solution to between about 6.7 andabout 7.1 immediately after or during the addition of the precipitatingalcohol. In another embodiment, the process improvement is realized bymaintaining the pH of the solution to between about 6.7 and about 7.1continuously during the precipitation incubation period. In otherembodiments, the pH of the solution is adjusted to between about 6.8 andabout 7.0 immediately after or during the addition of the precipitatingalcohol, or to a pH of about 6.7, 6.8, 6.9, 7.0, or 7.1 immediatelyafter or during the addition of the precipitating alcohol. In aparticular embodiment, the pH of the solution is adjusted to about 6.9immediately after or during the addition of the precipitating alcohol.In certain embodiments, the pH of the solution is maintained at betweenabout 6.8 to about 7.0 continuously during the precipitation incubationperiod, or at a pH of about 6.9 continuously during the precipitationincubation period. As such, in certain embodiments, a reduced amount ofIgG is lost in the supernatant fraction of the second precipitation stepas compared to an analogous precipitation step in which the pH of thesolution is adjusted prior to but not after addition of theprecipitating alcohol or to an analogous precipitation step in which thepH of the solution is not maintained during the entirety of theprecipitation incubation period. In one embodiment, the pH is maintainedat the desired pH during the precipitation hold or incubation time bycontinuously adjusting the pH of the solution. In one embodiment, thealcohol is ethanol.

In another embodiment, the process improvement is realized by adding theprecipitating alcohol and/or the solution used to adjust the pH byspraying, rather than by fluent addition. As such, in certainembodiments, a reduced amount of IgG is lost in the supernatant fractionof the second precipitation step as compared to an analogousprecipitation step in which the alcohol and/or solution used to adjustthe pH is introduced by fluent addition. In one embodiment, the alcoholis ethanol.

In another embodiment, the process improvement is realized by performingthe precipitation step at a temperature between about −7° C. and about−9° C. In one embodiment, the precipitation step is performed at atemperature of at or about −7° C. In another embodiment, theprecipitation step is performed at a temperature of at or about −8° C.In another embodiment, the precipitation step is performed at atemperature of at or about −9° C. In certain embodiments, the alcoholconcentration of the precipitation step is between about 23% and about27%. In a preferred embodiment, the alcohol concentration is betweenabout 24% and about 26%. In another preferred embodiment, the alcoholconcentration is at or about 25%. In other embodiments, the alcoholconcentration may be at or about 23%, 24%, 25%, 26%, or 27%. In aparticular embodiment, the second precipitation step is performed at atemperature of at or about −7° C. with an alcohol concentration of at orabout 25%. In one embodiment, the alcohol is ethanol.

The effect of increasing the alcohol concentration of the secondprecipitation from 20%, as used in Oncley et al., supra, to 25% andlowering the temperature of the incubation from −5° C., as used in theCohn and Oncley methods, to at or about −7° C. is a 5% to 6% increase inthe IgG content of the modified Fraction II+III precipitate.

In another embodiment, the process improvement is realized by adjustingthe pH of the solution to between about 6.7 and about 7.1, preferably ator about 6.9, immediately after or during the addition of theprecipitating alcohol, maintaining the pH of the solution at a pH ofbetween about 6.7 and about 7.1, preferably at or about 6.9, bycontinuously adjusting the pH during the precipitation incubationperiod, and by adding the precipitating alcohol and/or the solution usedto adjust the pH by spraying, rather than by fluent addition. In anotherparticular embodiment, the process improvement is realized by performingthe precipitation step at a temperature between about −7° C. and about−9° C., preferably at or about −7° C. and by precipitating the IgG withan alcohol concentration of between about 23% and about 27%, preferablyat or about 25%. In yet another particular embodiment, the processimprovement is realized by incorporating all of the Modified FractionII+III improvements provided above. In a preferred embodiment, theprocess improvement is realized by precipitating IgG at a temperature ofat or about −7° C. with at or about 25% ethanol added by spraying andthen adjusting the pH of the solution to at or about 6.9 after additionof the precipitating alcohol. In yet another preferred embodiment, thepH of the solution is maintained at or about 6.9 for the entirety of theprecipitation incubation or hold time.

4. Extraction of the Modified Fraction II+III Precipitate

In order to solubilize the IgG content of the modified Fraction II+IIIprecipitate, a cold extraction buffer is used to re-suspend theFractionation II+III precipitate at a typical ratio of 1 partprecipitate to 15 parts of extraction buffer. Other suitablere-suspension ratios may be used, for example from about 1:8 to about1:30, or from about 1:10 to about 1:20, or from about 1:12 to about1:18, or from about 1:13 to about 1:17, or from about 1:14 to about1:16. In certain embodiments, the re-suspension ratio may be about 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, or higher.

Suitable solutions for the extraction of the modified II+III precipitatewill generally have a pH between about 4.0 and about 5.5. In certainembodiments, the solution will have a pH between about 4.5 and about5.0, in other embodiments, the extraction solution will have a pH ofabout 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,5.3, 5.4, or 5.5. In a preferred embodiment, the pH of the extractionbuffer will be at or about 4.5. In another preferred embodiment, the pHof the extraction buffer will be at or about 4.7. In another preferredembodiment, the pH of the extraction buffer will be at or about 4.9.Generally, these pH requirements can be met using a buffering agentselected from, for example, acetate, citrate, monobasic phosphate,dibasic phosphate, mixtures thereof, and the like. Suitable bufferconcentrations typically range from about 5 to about 100 mM, or fromabout 10 to about 50 mM, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM buffering agent.

The extraction buffer will preferably have a conductivity of from about0.5 mS·cm⁻¹ to about 2.0 mS·cm⁻¹. For example, in certain embodiments,the conductivity of the extraction buffer will be about 0.5 mS·cm⁻¹, orabout 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, or about 2.0 mS·cm⁻¹. One of ordinary skill in the art will knowhow to generate extraction buffers having an appropriate conductivity.

In one particular embodiment, an exemplary extraction buffer may containat or about 5 mM monobasic sodium phosphate and at or about 5 mM acetateat a pH of at or about 4.5±0.2 and conductivity of at or about 0.7 to0.9 mS/cm.

Generally, the extraction is performed at between about 0° C. and about10° C., or between about 2° C. and about 8° C. In certain embodiments,the extraction may be performed at about 0° C., 1° C., 2° C., 3° C., 4°C., 5° C., 6° C., 7° C., 8° C., 9° C., or 10° C. In a particularembodiment, the extraction is performed at between about 2° C. and about10° C. Typically, the extraction process will proceed for between about60 and about 300 minutes, or for between about 120 and 240 min, or forbetween about 150 and 210 minutes, while the suspension is continuouslystirred. In certain embodiments, the extraction process will proceed forabout 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or about 300 minutes.In a preferred embodiment, the extraction process will proceed for atleast 160 minutes with continuous stirring.

It has been found that employing an extraction buffer containing 5 mMmonobasic sodium phosphate, 5 mM acetate, and 0.051% to 0.06% glacialacetic acid (v/v), a substantial increase in the yield increase in thefinal IgG composition can be obtained without jeopardizing the purity ofthe final product. The correlation of amount of acetic acid andextraction buffer pH is demonstrated in FIG. 9. In a preferredembodiment, the Fraction II+III precipitate is extracted with a paste tobuffer ration of at or about 1:15 at a pH of at or about 4.5±0.2.

Advantageously, it has been found that compared to the currentmanufacturing process for GAMMAGARD® LIQUID (Baxter Healthcare), whichemploys an extraction buffer containing 5 mM monobasic sodium phosphate,5 mM acetate, and 0.051% glacial acetic acid (v/v), that by increasingthe glacial acetic acid content to at or about 0.06% (v/v), asubstantial increase in the yield increase in the final IgG compositioncan be obtained. As compared to methods previously employed for theextraction of the precipitate formed by the second precipitation step(GAMMAGARD® LIQUID), the present invention provides, in severalembodiments, methods that result in improved IgG yields in the ModifiedFraction II+III suspension.

In one aspect, the improvement relates to a method in which a reducedamount of IgG is lost in the non-solubilized fraction of the ModifiedFraction II+III precipitate. In one embodiment, the process improvementis realized by extracting the Modified Fraction II+III precipitate at aratio of 1:15 (precipitate to buffer) with a solution containing 5 mMmonobasic sodium phosphate, 5 mM acetate, and 0.06% glacial acetic acid(v/v). In another embodiment, the improvement is realized by maintainingthe pH of the solution during the duration of the extraction process. Inone embodiment, the pH of the solution is maintained at between about4.1 and about 4.9 for the duration of the extraction process. In apreferred embodiment, the pH of the solution is maintained at betweenabout 4.2 and about 4.8 for the duration of the extraction process. In amore preferred embodiment, the pH of the solution is maintained atbetween about 4.3 and about 4.7 for the duration of the extractionprocess. In another preferred embodiment, the pH of the solution ismaintained at between about 4.4 and about 4.6 for the duration of theextraction process. In yet another preferred embodiment, the pH of thesolution is maintained at or at about 4.5 for the duration of theextraction process.

In another aspect, the improvement relates to a method in which anincreased amount of IgG is solubilized from the Fraction II+IIIprecipitate in the Fraction II+III dissolution step. In one embodiment,the process improvement is realized by solubilizing the Fraction II+IIIprecipitate in a dissolution buffer containing 600 mL glacial aceticacid per 1000 L. In another embodiment, the improvement relates to amethod in which impurities are reduced after the IgG in the FractionII+III precipitate is solubilized. In one embodiment, the processimprovement is realized by mixing finely divided silicon dioxide (SiO₂)with the Fraction II+III suspension for at least about 30 minutes.

5. Pretreatment and Filtration of the Modified Fraction II+IIISuspension

In order to remove the non-solubilized fraction of the Modified FractionII+III precipitate (i.e., the Modified Fraction II+III filter cake), thesuspension is filtered, typically using depth filtration. Depth filtersthat may be employed in the methods provided herein include, metallic,glass, ceramic, organic (such as diatomaceous earth) depth filters, andthe like. Example of suitable filters include, without limitation, Cuno50SA, Cuno 90SA, and Cuno VR06 filters (Cuno). Alternatively, theseparation step can be performed by centrifugation rather thanfiltration.

Although the manufacturing process improvements described above minimizeIgG losses in the initial steps of the purification process, criticalimpurities, including PKA activity, amidolytic activity, and fibrinogencontent, are much higher when, for example, the II+III paste isextracted at pH 4.5 or 4.6, as compared to when the extraction occurs ata pH around 4.9 to 5.0 (see, Examples 2 to 5).

In order to counter act the impurities extracted in the methods providedherein, it has now been found that the purity of the IgG composition canbe greatly enhanced by the addition of a pretreatment step prior tofiltration/centrifugation. In one embodiment, this pretreatment stepcomprises addition of finely divided silica dioxide particles (e.g.,fumed silica, Aerosil®) followed by a 40 to 80 minute incubation periodduring which the suspension is constantly mixed. In certain embodiments,the incubation period will be between about 50 minutes and about 70minutes, or about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or moreminutes. Generally, the treatment will be performed at between about 0°C. and about 10° C., or between about 2° C. and about 8° C. In certainembodiments, the treatment may be performed at about 0° C., 1° C., 2°C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., or 10° C. In aparticular embodiment, the treatment is performed at between about 2° C.and about 10° C.

The effect of the fumed silica treatment is exemplified by the resultsfound in Example 17. In this example, a Fraction II+III precipitate issuspended and split into two samples, one of which is clarified withfilter aid only prior to filtration (FIG. 7A) and one of which istreated with fumed silica prior to addition of the filter aid andfiltration (FIG. 7B). As can be seen in the chromatographs and in thequantitated data, the filtrate sample pretreated with fumed silica had amuch higher IgG purity than the sample only treated with filter aid(68.8% vs. 55.7%; compare Tables 17 and 18, respectively).

In certain embodiments, fumed silica is added at a concentration ofbetween about 20 g/kg II+III paste and about 100 g/kg II+III paste(i.e., for a Modified Fraction II+III precipitate that is extracted at aratio of 1:15, fumed silica should be added at a concentration fromabout 20 g/16 kg II+III suspension to about 100 g/16 kg II+IIIsuspension, or at a final concentration of about 0.125% (w/w) to about0.625% (w/w)). In certain embodiments, the fumed silica may be added ata concentration of about 20 g/kg II+III paste, or about 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 g/kg II+III paste. Inone specific embodiment, fumed silica (e.g., Aerosil 380 or equivalent)is added to the Modified Fraction II+III suspension to a finalconcentration of about 40 g/16 kg II+III. Mixing takes place at about 2to 8° C. for at least 50 to 70 minutes.

In certain embodiments, SiO₂ is added to a an IgG composition at aconcentration between about 0.01 g/g protein and about 10 g/g protein.In another embodiment, SiO₂ is added to a an IgG composition at aconcentration between about 0.01 g/g protein and about 5 g/g protein. Inanother embodiment, SiO₂ is added to an IgG composition at aconcentration between about 0.02 g/g protein and about 4 g/g protein. Inone embodiment, SiO₂ is added at a final concentration of at least 0.1 gper gram total protein. In another specific embodiment, fumed silica isadded at a concentration of at least 0.2 g per gram total protein. Inanother specific embodiment, fumed silica is added at a concentration ofat least 0.25 g per gram total protein. In other specific embodiments,fumed silica is added at a concentration of at least 1 g per gram totalprotein. In another specific embodiment, fumed silica is added at aconcentration of at least 2 g per gram total protein. In anotherspecific embodiment, fumed silica is added at a concentration of atleast 2.5 g per gram total protein. In yet other specific embodiments,finely divided silicon dioxide is added at a concentration of at least0.01 g/g total protein or at least 0.02 g, 0.03 g, 0.04 g, 0.05 g, 0.06g, 0.07 g, 0.08 g, 0.09 g, 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7g, 0.8 g, 0.9 g, 1.0 g, 1.5 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g, 4.0 g, 4.5 g,5.0 g, 5.5 g, 6.0 g, 6.5 g, 7.0 g, 7.5 g, 8.0 g, 8.5 g, 9.0 g, 9.5 g,10.0 g, or more per gram total protein.

In certain embodiments, filter aid, for example Celpure C300 (Celpure)or Hyflo-Supper-Cel (World Minerals), will be added after the silicadioxide treatment, to facilitate depth filtration. Filter aid can beadded at a final concentration of from about 0.01 kg/kg II+III paste toabout 1.0 kg/kg II+III paste, or from about 0.02 kg/kg II+III paste toabout 0.8 kg/kg II+III paste, or from about 0.03 kg/kg II+III paste toabout 0.7 kg/kg II+III paste. In other embodiments, filter aid can beadded at a final concentration of from about 0.01 kg/kg II+III paste toabout 0.07 kg/kg II+III paste, or from about 0.02 kg/kg II+III paste toabout 0.06 kg/kg II+III paste, or from about 0.03 kg/kg II+III paste toabout 0.05 kg/kg II+III paste. In certain embodiments, the filter aidwill be added at a final concentration of about 0.01 kg/kg II+III paste,or about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 kg/kg II+III paste.

A significant fraction of IgG was being lost during the filtration stepof the GAMMAGARD® LIQUID manufacturing process. It was found that thecurrent methods of post-filtration wash, using 1.8 dead volumes ofsuspension buffer to purge the filter press frames and lines, wereinsufficient for maximal recovery of IgG at this step. Surprisingly, itwas found that at least 3.0 dead volumes, preferably 3.6 dead volumes,of suspension buffer were required in order for efficient recovery oftotal IgG in the Modified Fraction II+III clarified suspension (see,Example 12 and FIG. 1). In certain embodiments, the filter press may bewashed with any suitable suspension buffer. In a particular embodiment,the wash buffer will comprise, for example, 5 mM monobasic sodiumphosphate, 5 mM acetate, and 0.015% glacial acetic acid (v/v).

In one aspect, the improvement relates to a method in which a reducedamount of IgG is lost during the Fraction II+III suspension filtrationstep. In one embodiment, the process improvement is realized bypost-washing the filter with at least about 3.6 dead volumes ofdissolution buffer containing 150 mL glacial acetic acid per 1000 L. Therelationship between the amount of glacial acetic acid and pH in thepost-wash buffer is shown in FIG. 10. In one embodiment, the pH of thepost-wash extraction buffer is between about 4.6 and about 5.3. In apreferred embodiment, the pH of the post-wash buffer is between about4.7 and about 5.2. In another preferred embodiment, the pH of thepost-wash buffer is between about 4.8 and about 5.1. In yet anotherpreferred embodiment, the pH of the post-wash buffer is between about4.9 and about 5.0.

As compared to methods previously employed for the clarification of thesuspension formed from the second precipitation step (GAMMAGARD®LIQUID), the present invention provides, in several embodiments, methodsthat result in improved IgG yields and purity in the clarified FractionII+III suspension. In one aspect, the improvement relates to a method inwhich a reduced amount of IgG is lost in the Modified Fraction II+IIIfilter cake. In other aspect, the improvement relates to a method inwhich a reduced amount of an impurity is found in the clarified FractionII+III suspension.

In one embodiment, the process improvements are realized by inclusion ofa fumed silica treatment prior to filtration or centrifugalclarification of a Fraction II+III suspension. In certain embodiments,the fumed silica treatment will include addition of from about 0.01kg/kg II+III paste to about 0.07 kg/kg II+III paste, or from about 0.02kg/kg II+III paste to about 0.06 kg/kg II+III paste, or from about 0.03kg/kg II+III paste to about 0.05 kg/kg II+III paste, or about 0.02 kg/kgII+III paste, 0.03 kg/kg II+III paste, 0.04 kg/kg II+III paste, 0.05kg/kg II+III paste, 0.06 kg/kg II+III paste, 0.07 kg/kg II+III paste,0.08 kg/kg II+III paste, 0.09 kg/kg II+III paste, or 0.1 kg/kg II+IIIpaste, and the mixture will be incubated for between about 50 minutesand about 70 minutes, or about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, or more minutes at a temperature between about 2° C. and about 8° C.In another embodiment, the process improvements are realized byinclusion of a fumed silica treatment which reduced the levels ofresidual fibrinogen, amidolytic activity, and/or prekallikrein activatoractivity. In a specific embodiment, the process improvements arerealized by inclusion of a fumed silica treatment, which reduces thelevels of FXI, FXIa, FXII, and FXIIa in the immunoglobulin preparation.

In another embodiment, the process improvements are realized by washingthe depth filter with between about 3 and about 5 volumes of the filterdead volume after completing the Modified Fraction II+III suspensionfiltration step. In certain embodiments, the filter will be washed withbetween about 3.5 volumes and about 4.5 volumes, or at least about 2.5,2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 volumes of thefilter dead volume. In a particular embodiment, the filter press will bewashed with at least about 3.6 dead volumes of suspension buffer.

6. Detergent Treatment

In order to remove additional contaminants from the Modified FractionII+III filtrate, the sample is next subjected to a detergent treatment.Methods for the detergent treatment of plasma derived fractions are wellknown in the art. Generally, any standard non-ionic detergent treatmentmay be used in conjunction with the methods provided herein. Forexample, an exemplary protocol for a detergent treatment is providedbelow.

Briefly, polysorbate-80 is added to the Modified Fraction II+IIIfiltrate at a final concentration of about 0.2% (w/v) with stirring andthe sample is incubated for at least 30 minutes at a temperature betweenabout 2 to 8° C. Sodium citrate dehydrate is then mixed into thesolution at a final concentration of about 8 g/L and the sample isincubated for an additional 30 minutes, with continuous of stirring at atemperature between about 2 to 8° C.

In certain embodiments, any suitable non-ionic detergent can be used.Examples of suitable non-ionic detergents include, without limitation,Octylglucoside, Digitonin, C12E8, Lubrol, Triton X-100, Nonidet P-40,Tween-20 (i.e., polysorbate-20), Tween-80 (i.e., polysorbate-80), analkyl poly(ethylene oxide), a Brij detergent, an alkylphenolpoly(ethylene oxide), a poloxamer, octyl glucoside, decyl maltoside, andthe like.

In one embodiment, a process improvement is realized by adding thedetergent reagents (e.g., polysorbate-80 and sodium citrate dehydrate)by spraying rather than by fluent addition. In other embodiments, thedetergent reagents may be added as solids to the Modified FractionII+III filtrate while the sample is being mixed to ensure rapiddistribution of the additives. In certain embodiments, it is preferableto add solid reagents by sprinkling the solids over a delocalizedsurface area of the filtrate such that local overconcentration does notoccur, such as in fluent addition.

7. Third Precipitation Event—Precipitation G

In order to remove several residual small proteins, such as albumin andtransferrin, a third precipitation is performed at a concentration of25% alcohol. Briefly, the pH of the detergent treated II+III filtrate isadjusted to between about 6.8 and 7.2, preferably between about 6.9 andabout 7.1, most preferably about 7.0 with a suitable pH modifyingsolution (e.g., 1M sodium hydroxide or 1M acetic acid). Cold alcohol isthen added to the solution to a final concentration of about 25% (v/v)and the mixture is incubated while stirring at between about −6° C. toabout −10° C. for at least 1 hour to form a third precipitate (i.e.,precipitate G). In one embodiment, the mixture is incubated for at lease2 hours, or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or more hours. In a preferredembodiment, the mixture is incubated for at least 2 hours. In a morepreferred embodiment, the mixture is incubated for at least 4 hours. Inan even more preferred embodiment, the mixture is incubated for at least8 hours.

In one aspect, a process improvement relates to a method in which areduced amount of IgG is lost in the supernatant fraction of the thirdprecipitation step. In certain embodiments, the process improvement isrealized by adjusting the pH of the solution to between about 6.8 andabout 7.2 immediately after or during the addition of the precipitatingalcohol. In another embodiment, the process improvement is realized bymaintaining the pH of the solution to between about 6.8 and about 7.2continuously during the precipitation incubation period. In otherembodiments, the pH of the solution is adjusted to between about 6.9 andabout 7.1 immediately after or during the addition of the precipitatingalcohol, or to a pH of about 6.8, 6.9, 7.0, 7.1, or 7.2 immediatelyafter or during the addition of the precipitating alcohol. In aparticular embodiment, the pH of the solution is adjusted to about 7.0immediately after or during the addition of the precipitating alcohol.In certain embodiments, the pH of the solution is maintained at betweenabout 6.9 to about 7.1 continuously during the precipitation incubationperiod, or at a pH of about 7.0 continuously during the precipitationincubation period. As such, in certain embodiments, a reduced amount ofIgG is lost in the supernatant fraction of the third precipitation stepas compared to an analogous precipitation step in which the pH of thesolution is adjusted prior to but not after addition of theprecipitating alcohol or to an analogous precipitation step in which thepH of the solution is not maintained during the entirety of theprecipitation incubation period. In one embodiment, the pH is maintainedat the desired pH during the precipitation hold or incubation time bycontinuously adjusting the pH of the solution. In one embodiment, thealcohol is ethanol.

In another embodiment, the process improvement is realized by adding theprecipitating alcohol and/or the solution used to adjust the pH byspraying, rather than by fluent addition. As such, in certainembodiments, a reduced amount of IgG is lost in the supernatant fractionof the third precipitation step as compared to an analogousprecipitation step in which the alcohol and/or solution used to adjustthe pH is introduced by fluent addition. In one embodiment, the alcoholis ethanol.

8. Suspension and Filtration of Precipitate G (PptG)

In order to solubilize the IgG content of the precipitate G, a coldextraction buffer is used to re-suspend the PptG. Briefly, theprecipitate G is dissolved 1 to 3.5 in Water for Injection (WFI) atbetween about 0° C. and about 8° C. to achieve an AU₂₈₀₋₃₂₀ value ofbetween about 40 to 95. The final pH of the solution, which is stirredfor at least 2 hours, is then adjusted to at or about 5.2±0.2. In oneembodiment, this pH adjustment is performed with 1M acetic acid. Toincrease the solubility of IgG, the conductivity of the suspension isincreased to between about 2.5 and about 6.0 mS/cm. In one embodiment,the conductivity is increased by the addition of sodium chloride. Thesuspended PptG solution is then filtered with a suitable depth filterhaving a nominal pore size of between about 0.1 μm and about 0.4 μm inorder to remove any undissolved particles. In one embodiment, thenominal pore size of the depth filter is about 0.2 μm (e.g., Cuno VR06filter or equivalent) to obtain a clarified filtrate. In anotherembodiment, the suspended PptG solution is centrifuged to recover aclarified supernatant. Post-wash of the filter is performed using asodium chloride solution with a conductivity of between about 2.5 andabout 6.0 mS/cm. Typically, suitable solutions for the extraction ofprecipitate G include, WFI and low conductivity buffers. In oneembodiment, a low conductivity buffer has a conductivity of less thanabout 10 mS/cm. In a preferred embodiment, the low conductivity bufferhas a conductivity of less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1mS/cm. In a preferred embodiment, the low conductivity buffer has aconductivity of less than about 6 mS/cm. In another preferredembodiment, the low conductivity buffer has a conductivity of less thanabout 4 mS/cm. In another preferred embodiment, the low conductivitybuffer has a conductivity of less than about 2 mS/cm.

9. Solvent Detergent Treatment

In order to inactivate various viral contaminants which may be presentin plasma-derived products, the clarified PptG filtrate is nextsubjected to a solvent detergent (S/D) treatment. Methods for thedetergent treatment of plasma derived fractions are well known in theart (for review see, Pelletier J P et al., Best Pract Res Clin Haematol.2006; 19(1):205-42). Generally, any standard S/D treatment may be usedin conjunction with the methods provided herein. For example, anexemplary protocol for an S/D treatment is provided below.

Briefly, Triton X-100, Tween-20, and tri(n-butyl)phosphate (TNBP) areadded to the clarified PptG filtrate at final concentrations of about1.0%, 0.3%, and 0.3%, respectively. The mixture is then stirred at atemperature between about 18° C. and about 25° C. for at least about anhour.

In one embodiment, a process improvement is realized by adding the S/Dreagents (e.g., Triton X-100, Tween-20, and TNBP) by spraying ratherthan by fluent addition. In other embodiments, the detergent reagentsmay be added as solids to the clarified PptG filtrate, which is beingmixed to ensure rapid distribution of the S/D components. In certainembodiments, it is preferable to add solid reagents by sprinkling thesolids over a delocalized surface area of the filtrate such that localoverconcentration does not occur, such as in fluent addition.

10. Ion Exchange Chromatography

In order to further purify and concentrate IgG from the S/D treated PptGfiltrate, cation exchange and/or anion exchange chromatography can beemployed. Methods for purifying and concentrating IgG using ion exchangechromatography are well known in the art. For example, U.S. Pat. No.5,886,154 describes a method in which a Fraction II+III precipitate isextracted at low pH (between about 3.8 and 4.5), followed byprecipitation of IgG using caprylic acid, and finally implementation oftwo anion exchange chromatography steps. U.S. Pat. No. 6,069,236describes a chromatographic IgG purification scheme that does not relyon alcohol precipitation at all. PCT Publication No. WO 2005/073252describes an IgG purification method involving the extraction of aFraction II+III precipitate, caprylic acid treatment, PEG treatment, anda single anion exchange chromatography step. U.S. Pat. No. 7,186,410describes an IgG purification method involving the extraction of eithera Fraction I+II+III or a Fraction II precipitate followed by a singleanion exchange step performed at an alkaline pH. U.S. Pat. No. 7,553,938describes a method involving the extraction of either a FractionI+II+III or a Fraction II+III precipitate, caprylate treatment, andeither one or two anion exchange chromatography steps. U.S. Pat. No.6,093,324 describes a purification method comprising the use of amacroporous anion exchange resin operated at a pH between about 6.0 andabout 6.6. U.S. Pat. No. 6,835,379 describes a purification method thatrelies on cation exchange chromatography in the absence of alcoholfractionation. The disclosures of the above publications are herebyincorporated by reference in their entireties for all purposes

In one embodiment of the methods of the present invention, the S/Dtreated PptG filtrate may be subjected to both cation exchangechromatography and anion exchange chromatography. For example, in oneembodiment, the S/D treated PptG filtrate is passed through a cationexchange column, which binds the IgG in the solution. The S/D reagentscan then be washed away from the absorbed IgG, which is subsequentlyeluted off of the column with a high pH elution buffer having a pHbetween about 8.0 and 9.0. In this fashion, the cation exchangechromatography step can be used to remove the S/D reagents from thepreparation, concentrate the IgG containing solution, or both. Incertain embodiments, the pH elution buffer may have a pH between about8.2 and about 8.8, or between about 8.4 and about 8.6, or a pH of about8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. In a preferredembodiment, the pH of the elution buffer is about 8.5±0.1.

In certain embodiments, the eluate from the cation exchange column maybe adjusted to a lower pH, for example between about 5.5 and about 6.5,and diluted with an appropriate buffer such that the conductivity of thesolution is reduced. In certain embodiments, the pH of the cationexchange eluate may be adjusted to a pH between about 5.7 and about 6.3,or between about 5.9 and about 6.1, or a pH of about 5.5, 5.6, 5.7, 5.8,5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In a preferred embodiment, the pHof the eluate is adjusted to a pH of about 6.0±0.1. The eluate is thenloaded onto an anion exchange column, which binds several contaminantsfound in the preparation. The column flow through, containing the IgGfraction, is collected during column loading and washing. In certainembodiments, the ion exchange chromatographic steps of the presentinvention can be performed in column mode, batch mode, or in acombination of the two.

In certain embodiments, a process improvement is realized by adding thesolution used to adjust the pH by spraying, rather than by fluentaddition.

11. Nanofiltration and Ultra/Diafiltration

In order to further reduce the viral load of the IgG compositionprovided herein, the anion exchange column effluent may be nanofilteredusing a suitable nanofiltration device. In certain embodiments, thenanofiltration device will have a mean pore size of between about 15 nmand about 200 nm. Examples of nanofilters suitable for this use include,without limitation, DVD, DV 50, DV 20 (Pall), Viresolve NFP, ViresolveNFR (Millipore), Planova 15N, 20N, 35N, and 75N (Planova). In a specificembodiment, the nanofilter may have a mean pore size of between about 15nm and about 72 nm, or between about 19 nm and about 35 nm, or of about15 nm, 19 nm, 35 nm, or 72 nm. In a preferred embodiment, the nanofilterwill have a mean pore size of about 35 nm, such as an Asahi PLANOVA 35Nfilter or equivalent thereof.

Optionally, ultrafiltration/diafiltration may performed to furtherconcentrate the nanofiltrate. In one embodiment, an open channelmembrane is used with a specifically designed post-wash and formulationnear the end the production process render the resulting IgGcompositions about twice as high in protein concentration (200 mg/mL)compared to state of the art IVIGs (e.g., GAMMAGARD® LIQUID) withoutaffecting yield and storage stability. With most of the commercialavailable ultrafiltration membranes a concentration of 200 mg/mL IgGcannot be reached without major protein losses. These membranes will beblocked early and therefore adequate post-wash is difficult to achieve.Therefore open channel membrane configurations have to be used. Evenwith open channel membranes, a specifically designed post-wash procedurehas to be used to obtain the required concentration without significantprotein loss (less than 2% loss). Even more surprising is the fact thatthe higher protein concentration of 200 mg/mL does not effect the virusinactivation capacity of the low pH storage step.

Subsequent to nanofiltration, the filtrate may be further concentratedby ultrafiltration/diafiltration. In one embodiment, the nanofiltratemay be concentrated by ultrafiltration to a protein concentration ofbetween about 2% and about 10% (w/v). In certain embodiments, theultrafiltration is carried out in a cassette with an open channel screenand the ultrafiltration membrane has a nominal molecular weight cut off(NMWCO) of less than about 100 kDa or less than about 90, 80, 70, 60,50, 40, 30, or fewer kDa. In a preferred embodiment, the ultrafiltrationmembrane has a NMWCO of no more than 50 kDa.

Upon completion of the ultrafiltration step, the concentrate may furtherbe concentrated via diafiltration against a solution suitable forintravenous or intramuscular administration. In certain embodiments, thediafiltration solution may comprise a stabilizing and/or bufferingagent. In a preferred embodiment, the stabilizing and buffering agent isglycine at an appropriate concentration, for example between about 0.20M and about 0.30M, or between about 0.22M and about 0.28M, or betweenabout 0.24M and about 0.26 mM, or at a concentration of about 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In a preferredembodiment, the diafiltration buffer contains at or about 0.25 Mglycine.

Typically, the minimum exchange volume is at least about 3 times theoriginal concentrate volume or at least about 4, 5, 6, 7, 8, 9, or moretimes the original concentrate volume. The IgG solution may beconcentrated to a final protein concentration of between about 5% andabout 25% (w/v), or between about 6% and about 18% (w/v), or betweenabout 7% and about 16% (w/v), or between about 8% and about 14% (w/v),or between about 9% and about 12%, or to a final concentration of about5%, or 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 21%, 22%, 23%, 24%, 25% or higher. In one embodiment, a finalprotein concentration of at least about 23% is achieved without addingthe post-wash fraction to the concentrated solution. In anotherembodiment, a final protein concentration of at least about 24% isachieved without adding the post-wash fraction to the concentratedsolution. a final protein concentration of at least about 25% isachieved without adding the post-wash fraction to the concentratedsolution. Typically, at the end of the concentration process, the pH ofthe solution will be between about 4.6 to 5.1.

In an exemplary embodiment, the pH of the IgG composition is adjusted toabout 4.5 prior to ultrafiltration. The solution is concentrated to aprotein concentration of 5±2% w/v through ultrafiltration. The UFmembrane has a nominal molecular weight cut off (NMWCO) of 50,000Daltons or less (Millipore Pellicon Polyether sulfon membrane). Theconcentrate is diafiltered against ten volumes of 0.25 M glycinesolution, pH 4.5±0.2. Throughout the ultra-diafiltration operation thesolution is maintained at a temperature of between about 2° C. to about8° C. After diafiltration, the solution is concentrated to a proteinconcentration of at least 11% (w/v).

12. Formulation

Upon completion of the diafiltration step, the protein concentration ofthe solution is adjusted to with the diafiltration buffer to a finalconcentration of between about 5% and about 20% (w/v), or between about6% and about 18% (w/v), or between about 7% and about 16% (w/v), orbetween about 8% and about 14% (w/v), or between about 9% and about 12%,or to a final concentration of about 5%, or 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In a preferredembodiment, the final protein concentration of the solution is betweenabout 9% and about 11%, more preferably about 10%.

The formulated bulk solution is further sterilized by filtering througha membrane filter with an absolute pore size of no more than about 0.22micron, for example about 0.2 micron. Then the solution is asepticallydispensed into final containers for proper sealing, with samples takenfor testing.

In one embodiment, the IgG composition is further adjusted to aconcentration of about 10.2±0.2% (w/v) with diafiltration buffer. The pHis adjusted to about 4.4 to about 4.9 if necessary. Finally, thesolution is sterile filtered and incubated for three weeks at or about30° C.

B. Factor H

In one embodiment, the present invention provides a method for reducingthe amount of a serine protease or a serine protease zymogen in aplasma-derived Factor H composition. In one specific embodiment, themethod comprises the steps of: (a) contacting the Factor H compositionwith finely divided silicon dioxide (SiO₂) under conditions suitable tobind at least one serine protease or serine protease zymogen; and (b)separating the SiO₂ from the Factor H composition to remove the boundserine protease or serine protease zymogen. In a preferred embodiment,the serine protease or serine protease zymogen is Factor XIa (FXIa),Factor XIIa (FXIIa), Factor XI (FXI), and/or Factor XII (FXII).

In one embodiment, the method further comprises the step of performing afirst Factor H protein enrichment step to form a first enriched Factor Hcomposition, prior to contacting the composition with finely dividedsilicon dioxide (SiO₂). In certain embodiments, the first Factor Hprotein enrichment step is selected from a protein precipitation step(e.g., an alcohol fractionation step), an ultrafiltration/diafiltrationstep, and a chromatographic step.

In certain embodiments, the methods described above further comprisesthe step of performing a second Factor H protein enrichment step to forma second enriched Factor H composition, prior to contacting thecomposition with finely divided silicon dioxide (SiO₂). In certainembodiments, the first Factor H protein enrichment step is selected froma protein precipitation step (e.g., an alcohol fractionation step), anultrafiltration/diafiltration step, and a chromatographic step.

Accordingly, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived Factor H composition, the method comprises the stepsof: (a) performing a first Factor H enrichment step to form a firstenriched plasma-derived Factor H composition; (b) performing a secondFactor H enrichment step to form a second enriched plasma-derived FactorH composition; (c) contacting the second enriched composition withfinely divided silicon dioxide (SiO₂) under conditions suitable to bindat least one serine protease or serine protease zymogen; and (d)separating the SiO₂ from the composition to remove the bound serineprotease or serine protease zymogen. In a preferred embodiment, theserine protease or serine protease zymogen is Factor XIa (FXIa), FactorXIIa (FXIIa), Factor XI (FXI), and/or Factor XII (FXII). In certainembodiments, the combination of first and second enrichment steps isselected from any one of variations Var. 1 to Var. 100, found in Table1.

In certain embodiments, the methods described above further comprisesthe step of performing an Factor H enrichment step after contacting thecomposition with finely divided silicon dioxide (SiO₂). In certainembodiments, the Factor H enrichment step is selected from a proteinprecipitation step (e.g., an alcohol fractionation step), anultrafiltration/diafiltration step, and a chromatographic step.

Accordingly, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived Factor H composition the method comprises the steps of:(a) performing a first Factor H enrichment step to form a first enrichedplasma-derived Factor H composition; (b) contacting the first enrichedcomposition with finely divided silicon dioxide (SiO₂) under conditionssuitable to bind at least one serine protease or serine proteasezymogen; (c) separating the SiO₂ from the composition to remove thebound serine protease or serine protease zymogen; and (d) performing asecond Factor H enrichment step to form a second enriched plasma-derivedFactor H composition. In a preferred embodiment, the serine protease orserine protease zymogen is Factor XIa (FXIa), Factor XIIa (FXIIa),Factor XI (FXI), and/or Factor XII (FXII). In certain embodiments, thecombination of first and second enrichment steps is selected from anyone of variations Var. 1 to Var. 100, found in Table 1.

Likewise, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived Factor H composition, the method comprising the stepsof: (a) performing a first Factor H enrichment step to form a firstenriched plasma-derived Factor H composition; (b) performing a secondFactor H enrichment step to form a second enriched plasma-derived FactorH composition; (c) contacting the second enriched composition withfinely divided silicon dioxide (SiO₂) under conditions suitable to bindat least one serine protease or serine protease zymogen; (d) separatingthe SiO₂ from the composition to remove the bound serine protease orserine protease zymogen; and (e) performing a third Factor H enrichmentstep to form a third enriched plasma-derived Factor H composition. In apreferred embodiment, the serine protease or serine protease zymogen isFactor XIa (FXIa), Factor XIIa (FXIIa), Factor XI (FXI), and/or FactorXII (FXII). In certain embodiments, the combination of first and secondenrichment steps is selected from any one of variations Var. 101 to Var.1100, found in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7,Table 8, Table 9, Table 10, or Table 11.

1. Methods for the Manufacture of Plasma-Derived Factor H

Regarding production, the claimed processes starting from human plasmashall be based on the sub-fractionation of typical industrialintermediates obtained by, e.g., the fractional precipitation by ethanolin the cold (reviewed in Schultze H E, Heremans J F; Molecular Biologyof Human Proteins. Volume I: Nature and Metabolism of ExtracellularProteins 1966, Elsevier Publishing Company; p. 236-317). A preferredembodiment of such purification is the purification of functional FactorH from side fractions of industrial scale plasma fractionation in such away that established and licensed manufacturing processes of plasmaproducts, which are under control of pharmaceutical regulatoryauthorities, like immunoglobulins, are not affected. For example, thefilter cake obtained after filtration of a Fraction II+III pastesuspension (Teschner W et al., Vox Sang. 2007 January; 92(1):42-55),Fraction I precipitate (Cohn et al., (1946) supra), Precipitate III(Schultze H E, Heremans J F; Molecular Biology of Human Proteins. VolumeI: Nature and Metabolism of Extracellular Proteins 1966, ElsevierPublishing Company; p. 236-317 at p. 253) and precipitate B (method ofKistler and Nitschmann; supra at p. 253) are examples of such industrialsources for Factor H. Starting from those side fractions, purificationprocedures known in the art can be used to purify Factor H. They may bebased on precipitation with polyethylene glycol (Nagasawa S, Stroud R M;Mol Immunol 1980; 17:1365-72), affinity chromatography via immobilizedheparin (citation as before), ion exchange chromatography (Crossley L G,Porter R R; Biochem J 1980; 191:173-82) and hydrophobic interactionchromatography (Ripoche J, Al Salihi A, Rousseaux J, Fontaine M; BiochemJ 1984; 221, 89-96).

In one embodiment, the starting material for the invention is preparedusing Cohn fractions. This fractionation is a well known fractionationused for the preparation of immunoglobulin preparations can be preparedfrom donor serum or monoclonal or recombinant immunoglobulins. In atypical example, blood is collected from healthy donors. Usually, theblood is collected from the same species of animal as the subject towhich the immunoglobulin preparation will be administered (typicallyreferred to as “homologous” immunoglobulins). The immunoglobulins areisolated from the blood by suitable procedures, such as, for example,Cohn fractionation, ultracentrifugation, electrophoretic preparation,ion exchange chromatography, affinity chromatography, immunoaffinitychromatography, polyethylene glycol fractionation, or the like. (See,e.g., Cohn et al., J. Am. Chem. Soc. 68:459-75 (1946); Oncley et al., J.Am. Chem. Soc. 71:541-50 (1949); Barundern et al., Vox Sang. 7:157-74(1962); Koblet et al., Vox Sang. 13:93-102 (1967); U.S. Pat. Nos.5,122,373 and 5,177,194; the disclosures of which are incorporatedherein by reference in their entireties for all purposes.) In oneembodiment, the present invention uses the discarded fractions from thepreparation of immunoglobulins. In a particular embodiment, the presentinvention uses the fraction that is found in a SiO₂ filtration cake oncethe Fraction II+III extract is filtered.

Generally, Factor H preparations according to the present invention canbe prepared from any suitable starting materials, for example, recoveredplasma or source plasma. In a typical example, blood or plasma iscollected from healthy donors. Usually, the blood is collected from thesame species of animal as the subject to which the Factor H preparationwill be administered (typically referred to as “homologous” Factor H).The Factor H is isolated from the blood or plasma by suitableprocedures, such as, for example, precipitation (alcohol fractionationor polyethylene glycol fractionation), chromatographic methods (ionexchange chromatography, affinity chromatography, immunoaffinitychromatography, etc.) ultracentrifugation, and electrophoreticpreparation, and the like. (See, e.g., Cohn et al., J. Am. Chem. Soc.68:459-75 (1946); Deutsch et al., J. Biol. Chem. 164:109-118; Oncley etal., J. Am. Chem. Soc. 71:541-50 (1949); Cohn et al., J. Am. Chem. Soc.72:465-474 (1950); Cohn et al., Blood Cells and Plasma Proteins: TheirState in Nature (J. L. Tullis, ed), pp. 1-58, Academic Press, New Yorkand London (1953); Nitschmann et al., Helv. Chim. Acta 37:866-873;Kistler and Nitschmann, Vox Sang. 7:414-424 (1962); Barundern et al.,Vox Sang. 7:157-74 (1962); Koblet et al., Vox Sang. 13:93-102 (1967);U.S. Pat. Nos. 5,122,373 and 5,177,194; the disclosures of which arehereby incorporated by reference in their entireties for all purposes).

In certain embodiments, Factor H is recovered from material otherwisediscarded during the manufacture of other commercially important bloodproducts by plasma fractionation. For example, in an exemplaryembodiment, Factor H is extracted from a Fraction I precipitate and/orextracted from a filter cake formed after centrifugation or filtrationof a re-suspended Fraction II+III paste. Advantageously, according tothe methods provided herein, industrial-scale preparation of Factor Hcan be achieved without the need for additional input plasma or theredesign and regulatory re-approval of existing manufacturing processesfor other commercially important plasma-derived blood products, such asIgG gamma globulins for intravenous (IVIG) or subcutaneousadministration.

In one aspect, the present invention provides a method for preparing anenriched Factor H composition having reduced serine protease and/orserine protease zymogen content from plasma by extracting Factor H froma plasma fraction and reducing the FXI, FXIa, FXII, and/or FXIIa contentwith a SiO₂ treatment method provided herein.

In one embodiment, a method is provided for preparing an enriched FactorH composition from plasma, the method comprising the steps of: (a)precipitating proteins from a cryo-poor plasma fraction, in a firstprecipitation step, with between about 6% and about 10% alcohol at a pHof between about 7.0 and about 7.5 to obtain a first precipitate and afirst supernatant; (b) precipitating Factor H from the firstsupernatant, in a second precipitation step, with between about 20% andabout 30% alcohol at a pH of between about 6.7 and about 7.3 to form asecond precipitate; (c) re-suspending the second precipitate to form asuspension; (d) mixing finely divided silicon dioxide (SiO₂) with thesuspension from step (c); (e) separating the suspension to form a filtercake and a supernatant; and (f) extracting Factor H from the SiO₂ filtercake under solution conditions that reduce the level of a serineprotease or serine protease zymogen in the final composition. In apreferred embodiment, the filter cake is separated from the supernatantby filtering the suspension through a filter press containing a suitablefilter. In one embodiment, Factor H can be extracted by re-circulatingan extraction buffer through a filter press containing a filter cake.

In a second aspect, the present invention provides a method forpreparing an enriched Factor H composition with reduced serine proteaseand/or serine protease zymogen content from plasma by extracting FactorH from a Fraction I precipitate.

In a preferred embodiment, a method is provided for preparing anenriched Factor H composition from plasma, the method comprising thesteps of: (a) precipitating proteins from a cryo-poor plasma fraction,in a first precipitation step, with between about 6% and about 10%alcohol at a pH of between about 7.0 and about 7.5 to obtain a firstprecipitate and a first supernatant; (b) extracting Factor H from theprecipitate with a Factor H extraction buffer, and (c) reducing thelevel of a serine protease or serine protease zymogen by treating thecomposition with SiO₂, using a suitable method provided herein.

In one aspect, a method is provided for preparing an enriched Factor Hcomposition from plasma, by extracting Factor H from a pool of two ormore manufacturing byproduct fractions created by a process designed toprovide a second blood protein, for example, IgG gamma globulins. In oneembodiment, the method comprises pooling a Fraction I precipitate and aFraction II+III filter cake formed during the manufacture of IgG gammaglobulins (e.g., IVIG) and extracting Factor H from the pooledfractions.

In certain embodiments, an enriched Factor H composition having reducedserine protease and/or serine protease zymogen content may be furtherpurified subsequent to extraction from a Fraction I precipitate and/orFraction II+III filter cake. Various methods are available for furtherpurifying Factor H, including without limitation, additionalprecipitation steps or fractionations, affinity chromatography, ionexchange chromatography, hydrophobic interaction chromatography, sizeexclusion chromatography, solvent/detergent (S/D) treatment,nanofiltration, ultrafiltration, diafiltration, and the like.

In one embodiment, the method further comprises precipitating impuritiesfrom an enriched Factor H composition. In certain embodiments, this stepcomprises precipitating at least one impurity, for example a lipid orprotein, from the composition and then separating the precipitate fromthe supernatant containing Factor H. Optionally, Factor H can then beprecipitated from the supernatant in a separate precipitation.

In a specific embodiment, a Factor H composition extracted from a plasmafraction (e.g., fraction I precipitate, fraction II+III precipitate,Precipitate B precipitate, etc.) is further enriched by precipitating atleast one impurity out of the solution using PEG at a finalconcentration of between about 2.5% and about 7.5%. In anotherembodiment, PEG is used at a final concentration of between about 3% andabout 7%. In another embodiment, PEG is used at a final concentration ofbetween about 4% and about 6%. In yet another embodiment, PEG is used ata final concentration of about 5%.

In another specific embodiment, a Factor H composition extracted from aplasma fraction (e.g., fraction I precipitate, fraction II+IIIprecipitate, Precipitate B precipitate, etc.) is further enriched byprecipitating Factor H out of the solution using PEG at a finalconcentration of between about 9% and about 15%. In another embodiment,PEG is used at a final concentration of between about 10% and about 14%.In another embodiment, PEG is used at a final concentration of betweenabout 11% and about 13%. In yet another embodiment, PEG is used at afinal concentration of about 12%.

In another specific embodiment, a Factor H composition extracted from aplasma fraction (e.g., fraction I precipitate, fraction II+IIIprecipitate, Precipitate B precipitate, etc.) is further enriched by (a)precipitating at least one impurity out of the solution; (b)precipitating Factor H out of the solution; and (c) recovering theprecipitate containing Factor H. In certain embodiments, theprecipitation steps are performed with alcohol (e.g., methanol orethanol), PEG, or a combination thereof. In a particular embodiment, theprecipitation steps are performed with PEG. In certain embodiments, thePEG concentration of the first precipitation step is between about 2.5%and about 7.5% and the PEG concentration of the second precipitationstep is between about 9% and about 15%. In a specific embodiment, thePEG concentration of the first step is between about 4% and about 6% andthe PEG concentration of the second step is between about 11% and about13%. In a more specific embodiment, the PEG concentration of the firstprecipitation step is about 5% and the PEG concentration of the secondprecipitation step is about 12%. In yet other embodiments, the PEGconcentration of the first and second precipitation steps is selectedfrom variations Var. 1101 and Var. 1221 listed in Table 16.

TABLE 16 PEG concentrations for enrichment of Factor H compositions. PEGConcentration - First Precipitation 2.5% 3.0% 3.5% 4.0% 4.5% 5.0% 5.5%6.0% 6.5% 7.0% 7.5% PEG Concentration - 2.5% Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. Second Precipitation 1101 1112 1123 11341145 1156 1167 1178 1189 1200 1211 3.0% Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 1102 1113 1124 1135 1146 1157 1168 1179 11901201 1212 3.5% Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.1103 1114 1125 1136 1147 1158 1169 1180 1191 1202 1213 4.0% Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 1104 1115 1126 1137 11481159 1170 1181 1192 1203 1214 4.5% Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 1105 1116 1127 1138 1149 1160 1171 1182 1193 12041215 5.0% Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 11061117 1128 1139 1150 1161 1172 1183 1194 1205 1216 5.5% Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 1107 1118 1129 1140 1151 11621173 1184 1195 1206 1217 6.0% Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. 1108 1119 1130 1141 1152 1163 1174 1185 1196 1207 12186.5% Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 1109 11201131 1142 1153 1164 1175 1186 1197 1208 1219 7.0% Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. 1110 1121 1132 1143 1154 1165 11761187 1198 1209 1220 7.5% Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. 1111 1122 1133 1144 1155 1166 1177 1188 1199 1210 1221

In certain embodiments, the method for preparing an enriched Factor Hcomposition further comprises at least one, preferably two,chromatographic steps to further enrich the purity of the composition.Generally, any suitable chromatographic method may be employed tofurther enrich the Factor H composition, for example, extracted from aFraction I precipitate or Fraction II+III filter cake. In certainembodiments, prior to chromatographic enrichment, the extracted Factor Hcomposition will be subjected one or more additional precipitationsteps, as described above, to reduce the impurities present in thecomposition, reduce the load volume for the chromatographic step, and/orexchange the buffer of the composition.

In certain embodiments, a Factor H composition may be further enrichedby a chromatographic step comprising anion exchange chromatography(AEC), cation exchange chromatography (CEC), heparin affinitychromatography, hydrophobic exchange chromatography (HIC),hydroxyapatite chromatography (HAP), immunoaffinity chromatography, sizeexclusion chromatography (i.e., gel filtration), or other suitablechromatographic step. Chromatographic steps may be performed in eitherbatch or column mode.

In a preferred embodiment, the method comprises the use of anionexchange chromatography and heparin affinity chromatography.

In certain embodiments, the methods provided herein for the preparationof an enriched Factor H composition will further include at least one,preferably at least two, most preferably at least three, viralinactivation or removal steps. Non-limiting examples of viralinactivation or removal steps that may be employed with the methodsprovided herein include, solvent detergent treatment (Horowitz et al.,Blood Coagul Fibrinolysis 1994 (5 Suppl 3):S21-S28 and Kreil et al.,Transfusion 2003 (43):1023-1028, both of which are herein expresslyincorporated by reference in their entirety for all purposes),nanofiltration (Hamamoto et al., Vox Sang 1989 (56)230-236 and Yuasa etal., J Gen Virol. 1991 (72 (pt 8)):2021-2024, both of which are hereinexpressly incorporated by reference in their entirety for all purposes),low pH incubation at high temperatures (Kempf et al., Transfusion 1991(31)423-427 and Louie et al., Biologicals 1994 (22):13-19), and heattreatment of lyophilized Factor H compositions (Piszkiewicz et al.,Thromb Res. 1987 Jul. 15; 47(2):235-41; Piszkiewicz et al., Curr StudHematol Blood Transfus. 1989; (56):44-54; Epstein and Fricke, ArchPathol Lab Med. 1990 March; 114(3):335-40).

In a preferred embodiment, the present invention provides a method ofpreparing a virally safe enriched Factor H composition having reducedserine protease and/or serine protease zymogen content comprising (i)extracting Factor H from a Fraction II+III filter cake using SiO₂, (ii)performing a first precipitation step to precipitate at least oneimpurity from the Factor H composition, (iii) performing a secondprecipitation step to precipitate Factor H from the composition, and(iv) performing at least one viral inactivation or removal step, therebypreparing a virally safe enriched Factor H composition. In oneembodiment, the precipitation steps comprise PEG precipitation. In aspecific embodiment, the PEG concentration of the first and secondprecipitation steps is selected from variations Var. 1101 and Var. 1221listed in Table 16.

2. Co-Binding and Differential Elution

In one aspect, the present invention provides a method for preparing aplasma-derived Factor H composition having a reduced amount of a serineprotease or a serine protease zymogen, the method comprisingco-extracting Factor H and a serine protease and/or serine proteasezymogen from a composition derived from pooled plasma by binding theproteins to finely divided silicon dioxide (SiO₂), eluting the serineprotease and/or serine protease zymogen from the SiO₂ under a firstsolution condition, and subsequently eluting Factor H from the SiO₂under a second solution condition. In a preferred embodiment, thestarting composition is a re-suspended Fraction II+III precipitate orequivalent precipitate thereof.

In a specific embodiment, the method comprises the steps of: (a)contacting a composition containing Factor H and at least one serineprotease or serine protease zymogen with finely divided silicon dioxide(SiO₂) under conditions suitable to bind the Factor H and at least oneserine protease or serine protease zymogen; (b) separating the SiO₂ fromthe composition; (c) eluting the serine protease or serine proteasezymogen from the SiO₂ under a solution condition in which the Factor Hremains bound; and (d) eluting the Factor H from the SiO₂.

In certain embodiments, a solution condition in which the Factor Hremains bound refers to a condition that preferentially elutes theserine protease or serine protease zymogen, while a substantial fractionof Factor H remains bound to the SiO₂. In one embodiment, a substantialfraction refers to at least 10% of the Factor H bound to the SiO₂. Inanother embodiment, a substantial fraction refers to at least 25% of theFactor H bound to the SiO₂. In another embodiment, a substantialfraction refers to at least 50% of the Factor H bound to the SiO₂. Inanother embodiment, a substantial fraction refers to at least 75% of theFactor H bound to the SiO₂. In yet other embodiments, a substantialfraction refers to at least 10% of the Factor H bound to the SiO₂, or atleast 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 96, 97, 98, 99, or more of the Factor H bound to the SiO₂.

In certain embodiments, differential elution of the serine protease orserine protease zymogen and Factor H is achieved by sequentiallycontacting (i.e., step-wise elution) the SiO₂ with a first solutioncondition (e.g., a first elution buffer) suitable to elute the majorityof the serine protease or serine protease zymogen but not a substantialfraction of the bound Factor H, and a second solution condition (e.g., asecond elution buffer) suitable to elute the substantial fraction ofbound Factor H from the SiO₂.

In other embodiments, differential elution of the serine protease orserine protease zymogen and Factor H is achieved by gradually changingthe solution conditions (i.e., with an elution gradient) from a firstsolution condition suitable to elute the majority of the serine proteaseor serine protease zymogen but not a substantial fraction of the boundFactor H to a second solution condition suitable to elute thesubstantial fraction of bound Factor H from the SiO₂. In this fashion,the serine protease or serine protease zymogen and Factor H contenteluted off of the SiO₂ may be partially overlapping. By fractionatingthe elution and characterizing the individual fractions, a Factor H poolmay be created from fractions having high Factor H content and lowserine protease or serine protease zymogen content.

Solution conditions that may be varied to achieve a desired result froma method described above include, without limitation, the pH of thesolution, the conductivity of the solution, the temperature of thesolution, the concentration of Factor H in the composition, and theconcentration of SiO₂ used in the method. Generally, suitable pH rangesfor methods of reducing serine protease and/or serine protease zymogencontent in a Factor H enriched composition range from about 3 to about11. Suitable conductivities for the methods described above range fromabout 0.1 mS/cm to about 100 mS/cm. Suitable temperatures for performingthe methods described above range from about −10° C. to about 90° C.Finely divided silicon dioxide may be used at a final concentrationranging from about 0.01 g/g protein to about 10 g/g protein. Finally,Factor H compositions may vary in concentration from about 0.001 mg/mLto about 100 mg/mL.

In one embodiment, the solution condition under which the serineprotease or serine protease zymogen is eluted from the SiO₂ and asignificant fraction of the Factor H remains bound comprises a pHbetween about 5.0 and about 11.0. In another embodiment, the pH isbetween about 6.0 and about 1.0. In another embodiment, the pH isbetween about 7.0 and about 9.0. In another embodiment, the pH isbetween about 7.5 and about 8.5. In yet another embodiment, the pH isbetween about 7.0 and about 8.0.

In a particular embodiment, the solution condition under which theserine protease or serine protease zymogen is eluted from the SiO₂ and asignificant fraction of the Factor H remains bound comprises a pH ofabout 7.0. In another specific embodiment, the pH is about 7.5. Inanother embodiment, the pH is about 8.0. In yet other embodiments, thepH is about 3.0 or about 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7,10.8, 10.9, or 11.0.

In one embodiment, the solution condition under which the serineprotease or serine protease zymogen is eluted from the SiO₂ and asignificant fraction of the Factor H remains bound comprises a pH of atleast 6.0. In another embodiment, the pH is at least 6.5. In anotherembodiment, the pH is at least 7.0. In yet another embodiment, the pH isat least 7.5. In yet other embodiments, the pH of the solution is atleast 3.0 or at least 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,8.5, 9.0, 9.5, 10.0, 10.5, or higher.

In another embodiment, of any of the methods described above, thesolution condition under which the serine protease or serine proteasezymogen is eluted from the SiO₂ and a significant fraction of the FactorH remains bound comprises a pH of no greater than about 11.0. In anotherembodiment, the pH is no greater about 10.0. In another embodiment, thepH is no greater about 9.0. In another embodiment, the pH is no greaterabout 8.0. In yet other embodiments, the pH is no greater than about11.0, or 10.5, 10.0, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0,4.5, 4.0, 3.5, or lower.

In one embodiment, the solution condition under which the serineprotease or serine protease zymogen is eluted from the SiO₂ and asignificant fraction of the Factor H remains bound comprises aconductivity of at least 10 mS/cm. In another embodiment, theconductivity is at least 20 mS/cm. In yet other embodiments, theconductivity of the solution condition is at least 2 mS/cm, or at least3 mS/cm, 4 mS/cm, 5 mS/cm, 6 mS/cm, 7 mS/cm, 8 mS/cm, 9 mS/cm, 10 mS/cm,11 mS/cm, 12 mS/cm, 13 mS/cm, 14 mS/cm, 15 mS/cm, 16 mS/cm, 17 mS/cm, 18mS/cm, 19 mS/cm, 20 mS/cm, 21 mS/cm, 22 mS/cm, 23 mS/cm, 24 mS/cm, 25mS/cm, 26 mS/cm, 27 mS/cm, 28 mS/cm, 29 mS/cm, 30 mS/cm, 31 mS/cm, 32mS/cm, 33 mS/cm, 34 mS/cm, 35 mS/cm, 36 mS/cm, 37 mS/cm, 38 mS/cm, 39mS/cm, 40 mS/cm, 41 mS/cm, 42 mS/cm, 43 mS/cm, 44 mS/cm, 45 mS/cm, 46mS/cm, 47 mS/cm, 48 mS/cm, 49 mS/cm, 50 mS/cm, 55 mS/cm, 60 mS/cm, 65mS/cm, 70 mS/cm, 75 mS/cm, 80 mS/cm, 85 mS/cm, 90 mS/cm, 95 mS/cm, 100mS/cm, or greater.

In one embodiment, the solution condition under which the serineprotease or serine protease zymogen is eluted from the SiO₂ and asignificant fraction of the Factor H remains bound comprises aconductivity between about 10 mS/cm and about 100 mS/cm. In anotherembodiment, the conductivity is between about 10 mS/cm and about 50mS/cm. In another embodiment, the conductivity is between about 20 mS/cmand about 100 mS/cm. In yet another embodiment, the conductivity isbetween about 20 mS/cm and about 50 mS/cm.

As shown in Example 5 and illustrated in FIG. 3, it was found that theuse of solution conditions having a pH greater than 6.0 (e.g., 7.5) andincreasing conductivity (e.g., greater than 6.0 mS/cm), results inincreased elution of serine proteases and/or serine protease zymogensfrom SiO₂, and decreased elution of Factor H from SiO₂. Advantageously,these findings can be used to provide methods for reducing the levels ofserine protease and serine protease zymogen present in Factor Hcompositions. In a particular embodiment of the methods described above,the solution condition under which the serine protease or serineprotease zymogen is eluted from the SiO₂ and a significant fraction ofthe Factor H remains bound comprises a conductivity of at least about 10mS/cm and a pH of at least 7.0. In another particular embodiment, thesolution condition comprises a conductivity of at least 10 mS/cm and apH of at least 7.5. In another embodiment, the solution conditioncomprises a conductivity of at least 20 mS/cm and a pH of at least 7.0.In yet another embodiment, the solution condition comprises aconductivity of at least 20 mS/cm and a pH of at least 7.5.

3. Co-Binding and Preferential Factor H Elution

In one aspect, the present invention provides a method for preparing aplasma-derived Factor H composition having a reduced amount of a serineprotease or a serine protease zymogen, the method comprisingco-extracting Factor H and a serine protease and/or serine proteasezymogen from a composition derived from pooled plasma by binding theproteins to finely divided silicon dioxide (SiO₂), and eluting theFactor H from the SiO₂ under conditions in which a substantial fractionof the bound serine protease and/or serine protease zymogen remainsbound to the SiO₂. In a preferred embodiment, the starting compositionis a re-suspended Fraction II+III precipitate or equivalent precipitatethereof.

In a specific embodiment, the method comprises the steps of: (a)contacting a composition containing Factor H and at least one serineprotease or serine protease zymogen with finely divided silicon dioxide(SiO₂) under conditions suitable to bind the Factor H and at least oneserine protease or serine protease zymogen; (b) separating the SiO₂ fromthe composition; and (c) eluting the Factor H from the SiO₂ under asolution condition in which the serine protease or serine proteasezymogen remains bound.

In certain embodiments, a solution condition in which the serineprotease or serine protease zymogen remains bound refers to a conditionthat preferentially elutes the Factor H, while a substantial fraction ofthe serine protease or serine protease zymogen remains bound to theSiO₂. In one embodiment, a substantial fraction refers to at least 10%of the serine protease or serine protease zymogen bound to the SiO₂. Inanother embodiment, a substantial fraction refers to at least 25% of theserine protease or serine protease zymogen bound to the SiO₂. In anotherembodiment, a substantial fraction refers to at least 50% of the serineprotease or serine protease zymogen bound to the SiO₂. In anotherembodiment, a substantial fraction refers to at least 75% of the serineprotease or serine protease zymogen bound to the SiO₂. In yet otherembodiments, a substantial fraction refers to at least 10% of the serineprotease or serine protease zymogen bound to the SiO₂, or at least 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or more of the serine protease or serine protease zymogen boundto the SiO₂.

Solution conditions that may be varied to achieve a desired result froma method described above include, without limitation, the pH of thesolution, the conductivity of the solution, the temperature of thesolution, the concentration of Factor H in the composition, and theconcentration of SiO₂ used in the method. Generally, suitable pH rangesfor methods of reducing serine protease and/or serine protease zymogencontent in a Factor H enriched composition range from about 3 to about11. Suitable conductivities for the methods described above range fromabout 0.1 mS/cm to about 100 mS/cm. Suitable temperatures for performingthe methods described above range from about −10° C. to about 90° C.Finely divided silicon dioxide may be used at a final concentrationranging from about 0.01 g/g protein to about 10 g/g protein. Finally,Factor H compositions may vary in concentration from about 0.001 mg/mLto about 100 mg/mL.

In one embodiment, the solution condition under which the Factor H iseluted from the SiO₂ and a significant fraction of the serine proteaseor serine protease zymogen remains bound comprises a pH between about5.0 and about 11.0. In another embodiment, the pH is between about 6.0and about 1.0. In another embodiment, the pH is between about 7.0 andabout 9.0. In another embodiment, the pH is between about 7.5 and about8.5. In yet another embodiment, the pH is between about 7.0 and about8.0.

In a particular embodiment, the solution condition under which theFactor H is eluted from the SiO₂ and a significant fraction of theserine protease or serine protease zymogen remains bound comprises a pHof about 7.0. In another specific embodiment, the pH is about 7.5. Inanother embodiment, the pH is about 8.0. In yet other embodiments, thepH is about 3.0 or about 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7,10.8, 10.9, or 11.0.

In one embodiment, the solution condition under which the Factor H iseluted from the SiO₂ and a significant fraction of the serine proteaseor serine protease zymogen remains bound comprises a pH of at least 6.0.In another embodiment, the pH is at least 6.5. In another embodiment,the pH is at least 7.0. In yet another embodiment, the pH is at least7.5. In yet other embodiments, the pH of the solution is at least 3.0 orat least 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0,9.5, 10.0, 10.5, or higher.

In another embodiment, of any of the methods described above, thesolution condition under which the Factor H is eluted from the SiO₂ anda significant fraction of the serine protease or serine protease zymogenremains bound comprises a pH of no greater than about 11.0. In anotherembodiment, the pH is no greater about 10.0. In another embodiment, thepH is no greater about 9.0. In another embodiment, the pH is no greaterabout 8.0. In yet other embodiments, the pH is no greater than about11.0, or 10.5, 10.0, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0,4.5, 4.0, 3.5, or lower.

In one embodiment, the solution condition under which the Factor H iseluted from the SiO₂ and a significant fraction of the serine proteaseor serine protease zymogen remains bound comprises a conductivity of nomore than about 20 mS/cm. In another embodiment, the conductivity is nomore than about 10 mS/cm. In yet other embodiments, the conductivity ofthe solution condition is no more than about 20 mS/cm, or no more thanabout 19 mS/cm, 18 mS/cm, 17 mS/cm, 16 mS/cm, 15 mS/cm, 14 mS/cm, 13mS/cm, 12 mS/cm, 11 mS/cm, 10 mS/cm, 9 mS/cm, 8 mS/cm, 7 mS/cm, 6 mS/cm,5 mS/cm, 4 mS/cm, 3 mS/cm, 2 mS/cm, or less.

In one embodiment, the solution condition under which the serineprotease or serine protease zymogen is eluted from the SiO₂ and asignificant fraction of the Factor H remains bound comprises aconductivity between about 2 mS/cm and about 20 mS/cm. In anotherembodiment, the conductivity is between about 2 mS/cm and about 10mS/cm. In another embodiment, the conductivity is between about 20 mS/cmand about 6 mS/cm. In yet another embodiment, the conductivity isbetween about 10 mS/cm and about 6 mS/cm.

As shown in Example 5 and illustrated in FIG. 3, it was found that theuse of solution conditions having a pH greater than 6.0 (e.g., 7.5) anddecreasing conductivity (e.g., less than 20 mS/cm), results in increasedelution of Factor H from SiO₂, and decreased elution of serine proteasesand/or serine protease zymogens from SiO₂. Advantageously, thesefindings can be used to provide methods for reducing the levels ofserine protease and serine protease zymogen present in Factor Hcompositions. In a particular embodiment of the methods described above,the solution condition under which the Factor H is eluted from the SiO₂and a significant fraction of the serine protease or serine proteasezymogen remains bound comprises a conductivity of at no more than about20 mS/cm and a pH of at least 7.0. In another particular embodiment, thesolution condition comprises a conductivity of no more than about 10mS/cm and a pH of at least 7.5. In another embodiment, the solutioncondition comprises a conductivity between about 10 mS/cm and about 2mS/cm and a pH of at least 7.0. In yet another embodiment, the solutioncondition comprises a conductivity between about 10 mS/cm and about 2mS/cm and a pH of at least 7.5.

4. Preferential Binding of Factor H

In one aspect, the present invention provides a method for preparing aplasma-derived Factor H composition having a reduced amount of a serineprotease or a serine protease zymogen, the method comprising (a)contacting a composition containing Factor H and at least one serineprotease or serine protease zymogen with finely divided silicon dioxide(SiO₂) under conditions suitable to bind the Factor H but not the atleast one serine protease or serine protease zymogen; (b) separating theSiO₂ from the composition; and (c) eluting the Factor H from the SiO₂.

In certain embodiments, a solution condition in which the serineprotease or serine protease zymogen does not bind to the SiO₂ refers toa condition that preferentially allows Factor H binding to the SiO₂,while a substantial fraction of the serine protease or serine proteasezymogen remains unbound in the solution. In one embodiment, asubstantial fraction refers to at least 10% of the serine protease orserine protease zymogen in the starting composition. In anotherembodiment, a substantial fraction refers to at least 25% of the serineprotease or serine protease zymogen in the starting composition. Inanother embodiment, a substantial fraction refers to at least 50% of theserine protease or serine protease zymogen in the starting composition.In another embodiment, a substantial fraction refers to at least 75% ofthe serine protease or serine protease zymogen in the startingcomposition. In yet other embodiments, a substantial fraction refers toat least 10% of the serine protease or serine protease zymogen in thestarting composition, or at least 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more of the serineprotease or serine protease zymogen in the starting composition.

Solution conditions that may be varied to achieve a desired result froma method described above include, without limitation, the pH of thesolution, the conductivity of the solution, the temperature of thesolution, the concentration of Factor H in the composition, and theconcentration of SiO₂ used in the method. Generally, suitable pH rangesfor methods of reducing serine protease and/or serine protease zymogencontent in a Factor H enriched composition range from about 3 to about11. Suitable conductivities for the methods described above range fromabout 0.1 mS/cm to about 100 mS/cm. Suitable temperatures for performingthe methods described above range from about −10° C. to about 90° C.Finely divided silicon dioxide may be used at a final concentrationranging from about 0.01 g/g protein to about 10 g/g protein. Finally,Factor H compositions may vary in concentration from about 0.001 mg/mLto about 100 mg/mL.

In one embodiment, the solution condition under which Factor H binds toSiO₂ and a significant fraction of the serine protease or serineprotease zymogen does not bind comprises a pH between about 5.0 andabout 11.0. In another embodiment, the pH is between about 6.0 and about1.0. In another embodiment, the pH is between about 7.0 and about 9.0.In another embodiment, the pH is between about 7.5 and about 8.5. In yetanother embodiment, the pH is between about 7.0 and about 8.0.

In a particular embodiment, the solution condition under which Factor Hbinds to SiO₂ and a significant fraction of the serine protease orserine protease zymogen does not bind comprises a pH of about 7.0. Inanother specific embodiment, the pH is about 7.5. In another embodiment,the pH is about 8.0. In yet other embodiments, the pH is about 3.0 orabout 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5,8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9,10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0.

In one embodiment, the solution condition under which Factor H binds toSiO₂ and a significant fraction of the serine protease or serineprotease zymogen does not bind comprises a pH of at least 6.0. Inanother embodiment, the pH is at least 6.5. In another embodiment, thepH is at least 7.0. In yet another embodiment, the pH is at least 7.5.In yet other embodiments, the pH of the solution is at least 3.0 or atleast 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5,10.0, 10.5, or higher.

In another embodiment, of any of the methods described above, thesolution condition under which Factor H binds to SiO₂ and a significantfraction of the serine protease or serine protease zymogen does not bindcomprises a pH of no greater than about 11.0. In another embodiment, thepH is no greater about 10.0. In another embodiment, the pH is no greaterabout 9.0. In another embodiment, the pH is no greater about 8.0. In yetother embodiments, the pH is no greater than about 11.0, or 10.5, 10.0,9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, orlower.

In one embodiment, the solution condition under which Factor H binds toSiO₂ and a significant fraction of the serine protease or serineprotease zymogen does not bind comprises a conductivity of at least 10mS/cm. In another embodiment, the conductivity is at least 20 mS/cm. Inyet other embodiments, the conductivity of the solution condition is atleast 2 mS/cm, or at least 3 mS/cm, 4 mS/cm, 5 mS/cm, 6 mS/cm, 7 mS/cm,8 mS/cm, 9 mS/cm, 10 mS/cm, 11 mS/cm, 12 mS/cm, 13 mS/cm, 14 mS/cm, 15mS/cm, 16 mS/cm, 17 mS/cm, 18 mS/cm, 19 mS/cm, 20 mS/cm, 21 mS/cm, 22mS/cm, 23 mS/cm, 24 mS/cm, 25 mS/cm, 26 mS/cm, 27 mS/cm, 28 mS/cm, 29mS/cm, 30 mS/cm, 31 mS/cm, 32 mS/cm, 33 mS/cm, 34 mS/cm, 35 mS/cm, 36mS/cm, 37 mS/cm, 38 mS/cm, 39 mS/cm, 40 mS/cm, 41 mS/cm, 42 mS/cm, 43mS/cm, 44 mS/cm, 45 mS/cm, 46 mS/cm, 47 mS/cm, 48 mS/cm, 49 mS/cm, 50mS/cm, 55 mS/cm, 60 mS/cm, 65 mS/cm, 70 mS/cm, 75 mS/cm, 80 mS/cm, 85mS/cm, 90 mS/cm, 95 mS/cm, 100 mS/cm, or greater.

In one embodiment, the solution condition under which Factor H binds toSiO₂ and a significant fraction of the serine protease or serineprotease zymogen does not bind comprises a conductivity between about 10mS/cm and about 100 mS/cm. In another embodiment, the conductivity isbetween about 10 mS/cm and about 50 mS/cm. In another embodiment, theconductivity is between about 20 mS/cm and about 100 mS/cm. In yetanother embodiment, the conductivity is between about 20 mS/cm and about50 mS/cm.

As shown in Example 5 and illustrated in FIG. 3, it was found that theuse of solution conditions having a pH greater than 6.0 (e.g., 7.5) andincreasing conductivity (e.g., greater than 6.0 mS/cm), results in adecreased affinity of serine proteases and/or serine protease zymogensfor SiO₂, and increased affinity of Factor H for SiO₂. Advantageously,these findings can be used to provide methods for reducing the levels ofserine protease and serine protease zymogen present in Factor Hcompositions. In a particular embodiment of the methods described above,the solution condition under which Factor H binds to SiO₂ and asignificant fraction of the serine protease or serine protease zymogendoes not bind comprises a conductivity of at least about 10 mS/cm and apH of at least 7.0. In another particular embodiment, the solutioncondition comprises a conductivity of at least 10 mS/cm and a pH of atleast 7.5. In another embodiment, the solution condition comprises aconductivity of at least 20 mS/cm and a pH of at least 7.0. In yetanother embodiment, the solution condition comprises a conductivity ofat least 20 mS/cm and a pH of at least 7.5.

5. Preferential binding of Serine Protease or Serine Protease Zymogen

In one aspect, the present invention provides a method for preparing aplasma-derived Factor H composition having a reduced amount of a serineprotease or a serine protease zymogen, the method comprising (a)contacting a composition containing Factor H and at least one serineprotease or serine protease zymogen with finely divided silicon dioxide(SiO₂) under conditions suitable to bind the serine protease and/orserine protease zymogen but not the Factor H; and (b) separating theSiO₂ from the composition.

In certain embodiments, a solution condition in which the Factor H doesnot bind to the SiO₂ refers to a condition that preferentially allowsserine protease or serine protease zymogen binding to the SiO₂, while asubstantial fraction of the Factor H remains unbound in the solution. Inone embodiment, a substantial fraction refers to at least 10% of theFactor H in the starting composition. In another embodiment, asubstantial fraction refers to at least 25% of the Factor H in thestarting composition. In another embodiment, a substantial fractionrefers to at least 50% of the Factor H in the starting composition. Inanother embodiment, a substantial fraction refers to at least 75% of theFactor H in the starting composition. In yet other embodiments, asubstantial fraction refers to at least 10% of the Factor H in thestarting composition, or at least 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more of the Factor Hin the starting composition.

Solution conditions that may be varied to achieve a desired result froma method described above include, without limitation, the pH of thesolution, the conductivity of the solution, the temperature of thesolution, the concentration of Factor H in the composition, and theconcentration of SiO₂ used in the method. Generally, suitable pH rangesfor methods of reducing serine protease and/or serine protease zymogencontent in a Factor H enriched composition range from about 3 to about11. Suitable conductivities for the methods described above range fromabout 0.1 mS/cm to about 100 mS/cm. Suitable temperatures for performingthe methods described above range from about −10° C. to about 90° C.Finely divided silicon dioxide may be used at a final concentrationranging from about 0.01 g/g protein to about 10 g/g protein. Finally,Factor H compositions may vary in concentration from about 0.001 mg/mLto about 100 mg/mL.

In one embodiment, the solution condition under which the serineprotease or serine protease zymogen binds to SiO₂ and a significantfraction of the Factor H does not bind comprises a pH between about 5.0and about 11.0. In another embodiment, the pH is between about 6.0 andabout 1.0. In another embodiment, the pH is between about 7.0 and about9.0. In another embodiment, the pH is between about 7.5 and about 8.5.In yet another embodiment, the pH is between about 7.0 and about 8.0.

In a particular embodiment, the solution condition under which theserine protease or serine protease zymogen binds to SiO₂ and asignificant fraction of the Factor H does not bind comprises a pH ofabout 7.0. In another specific embodiment, the pH is about 7.5. Inanother embodiment, the pH is about 8.0. In yet other embodiments, thepH is about 3.0 or about 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7,10.8, 10.9, or 11.0.

In one embodiment, the solution condition under which the serineprotease or serine protease zymogen binds to SiO₂ and a significantfraction of the Factor H does not bind comprises a pH of at least 6.0.In another embodiment, the pH is at least 6.5. In another embodiment,the pH is at least 7.0. In yet another embodiment, the pH is at least7.5. In yet other embodiments, the pH of the solution is at least 3.0 orat least 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0,9.5, 10.0, 10.5, or higher.

In another embodiment, of any of the methods described above, thesolution condition under which the serine protease or serine proteasezymogen binds to SiO₂ and a significant fraction of the Factor H doesnot bind comprises a pH of no greater than about 11.0. In anotherembodiment, the pH is no greater about 10.0. In another embodiment, thepH is no greater about 9.0. In another embodiment, the pH is no greaterabout 8.0. In yet other embodiments, the pH is no greater than about11.0, or 10.5, 10.0, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0,4.5, 4.0, 3.5, or lower.

In one embodiment, the solution condition under which the serineprotease or serine protease zymogen binds to SiO₂ and a significantfraction of the Factor H does not bind comprises a conductivity of nomore than about 20 mS/cm. In another embodiment, the conductivity is nomore than about 10 mS/cm. In yet other embodiments, the conductivity ofthe solution condition is no more than about 20 mS/cm, or no more thanabout 19 mS/cm, 18 mS/cm, 17 mS/cm, 16 mS/cm, 15 mS/cm, 14 mS/cm, 13mS/cm, 12 mS/cm, 11 mS/cm, 10 mS/cm, 9 mS/cm, 8 mS/cm, 7 mS/cm, 6 mS/cm,5 mS/cm, 4 mS/cm, 3 mS/cm, 2 mS/cm, or less.

In one embodiment, the solution condition under which the serineprotease or serine protease zymogen binds to SiO₂ and a significantfraction of the Factor H does not bind comprises a conductivity betweenabout 2 mS/cm and about 20 mS/cm. In another embodiment, theconductivity is between about 2 mS/cm and about 10 mS/cm. In anotherembodiment, the conductivity is between about 20 mS/cm and about 6mS/cm. In yet another embodiment, the conductivity is between about 10mS/cm and about 6 mS/cm.

As shown in Example 5 and illustrated in FIG. 3, it was found that theuse of solution conditions having a pH greater than 6.0 (e.g., 7.5) anddecreasing conductivity (e.g., less than 20 mS/cm), results in increasedaffinity of Factor H for SiO₂, and decreased affinity of serineproteases and/or serine protease zymogens from SiO₂. Advantageously,these findings can be used to provide methods for reducing the levels ofserine protease and serine protease zymogen present in Factor Hcompositions. In a particular embodiment of the methods described above,the solution condition under which the serine protease or serineprotease zymogen binds to SiO₂ and a significant fraction of the FactorH does not bind comprises a conductivity of at no more than about 20mS/cm and a pH of at least 7.0. In another particular embodiment, thesolution condition comprises a conductivity of no more than about 10mS/cm and a pH of at least 7.5. In another embodiment, the solutioncondition comprises a conductivity between about 10 mS/cm and about 2mS/cm and a pH of at least 7.0. In yet another embodiment, the solutioncondition comprises a conductivity between about 10 mS/cm and about 2mS/cm and a pH of at least 7.5.

6. Method for Factor H Extraction from a Plasma Precipitate

In one aspect, the present invention provides a method for preparing aFactor H composition, the method comprising the steps of: (a) contactinga suspended plasma precipitate composition containing Factor H and atleast one serine protease or serine protease zymogen with finely dividedsilicon dioxide (SiO₂) under conditions suitable to bind the Factor H,(b) washing the SiO₂ with a solution comprising a pH between 5.0 and 7.0and a conductivity of less than 4 mS/cm, and (c) eluting the Factor Hfrom the SiO₂ with a solution comprising a pH between 7.0 and 8.0 and aconductivity greater than 10 mS/cm, thereby providing an enriched FactorH composition. In a preferred embodiment, the serine protease or serineprotease zymogen is one or more of FXI, FXIa, FXII, and FXIIa. Incertain embodiments, the plasma precipitate is a Cohn fraction Iprecipitate, a Cohn fraction II+III precipitate, a Cohn fractionI+II+III precipitate, a Kistler/Nitschmann Precipitate A, aKistler/Nitschmann Precipitate B, or an equivalent fraction thereof. Inone embodiment, the solution used to wash the SiO₂ comprises a pHbetween 5.5 and 6.5. In a specific embodiment, the solution used to washthe SiO₂ comprises a pH of 6.0±0.2. In one embodiment, the solution usedto elute Factor H comprises a conductivity of at least 20 mS/cm. In aspecific embodiment, the solution used to elute Factor H comprises aconductivity of between 25 mS/cm and 40 mS/cm.

In certain embodiments, the method described above further comprises anenrichment step comprising precipitating at least one impurity from theenriched Factor H composition, wherein Factor H is not co-precipitated.In a specific embodiment, the method comprises the steps of (a)contacting a suspended plasma precipitate composition containing FactorH and at least one serine protease or serine protease zymogen withfinely divided silicon dioxide (SiO₂) under conditions suitable to bindthe Factor H, (b) washing the SiO₂ with a solution comprising a pHbetween 5.0 and 7.0 and a conductivity of less than 4 mS/cm, (c) elutingthe Factor H from the SiO₂ with a solution comprising a pH between 7.0and 8.0 and a conductivity greater than 10 mS/cm, and (d) precipitatingat least one impurity from the Factor H elution, wherein Factor H is notprecipitated, thereby providing an enriched Factor H composition. In apreferred embodiment, the serine protease or serine protease zymogen isone or more of FXI, FXIa, FXII, and FXIIa. In certain embodiments, theplasma precipitate is a Cohn fraction I precipitate, a Cohn fractionII+III precipitate, a Cohn fraction I+II+III precipitate, aKistler/Nitschmann Precipitate A, a Kistler/Nitschmann Precipitate B, oran equivalent fraction thereof. In one embodiment, the solution used towash the SiO₂ comprises a pH between 5.5 and 6.5. In a specificembodiment, the solution used to wash the SiO₂ comprises a pH of6.0±0.2. In one embodiment, the solution used to elute Factor Hcomprises a conductivity of at least 20 mS/cm. In a specific embodiment,the solution used to elute Factor H comprises a conductivity of between25 mS/cm and 40 mS/cm. In one embodiment, the impurity precipitationstep is PEG precipitation. In a specific embodiment, the impurity PEGprecipitation comprises precipitation with PEG 4000 at a finalconcentration between 3% and 7%. In a more specific embodiment, thefinal concentration of PEG 4000 in the impurity precipitation step is5±0.5%.

In certain embodiments, the methods described above further comprises anenrichment step comprising precipitating Factor H from an enrichedFactor H composition. In a specific embodiment, the method comprises thesteps of (a) contacting a suspended plasma precipitate compositioncontaining Factor H and at least one serine protease or serine proteasezymogen with finely divided silicon dioxide (SiO₂) under conditionssuitable to bind the Factor H, (b) washing the SiO₂ with a solutioncomprising a pH between 5.0 and 7.0 and a conductivity of less than 4mS/cm, (c) eluting the Factor H from the SiO₂ with a solution comprisinga pH between 7.0 and 8.0 and a conductivity greater than 10 mS/cm, (d)precipitating at least one impurity from the Factor H elution, to form asupernatant comprising Factor H, and (e) precipitating Factor H from thesupernatant, thereby providing an enriched Factor H composition. In apreferred embodiment, the serine protease or serine protease zymogen isone or more of FXI, FXIa, FXII, and FXIIa. In certain embodiments, theplasma precipitate is a Cohn fraction I precipitate, a Cohn fractionII+III precipitate, a Cohn fraction I+II+III precipitate, aKistler/Nitschmann Precipitate A, a Kistler/Nitschmann Precipitate B, oran equivalent fraction thereof. In one embodiment, the solution used towash the SiO₂ comprises a pH between 5.5 and 6.5. In a specificembodiment, the solution used to wash the SiO₂ comprises a pH of6.0±0.2. In one embodiment, the solution used to elute Factor Hcomprises a conductivity of at least 20 mS/cm. In a specific embodiment,the solution used to elute Factor H comprises a conductivity of between25 mS/cm and 40 mS/cm. In one embodiment, the impurity precipitationstep is PEG precipitation. In a specific embodiment, the impurity PEGprecipitation comprises precipitation with PEG 4000 at a finalconcentration between 3% and 7%. In a more specific embodiment, thefinal concentration of PEG 4000 in the impurity precipitation step is5±0.5%. In one embodiment, the Factor H precipitation step is PEGprecipitation. In a specific embodiment, the Factor H PEG precipitationcomprises precipitation with PEG 4000 at a final concentration between10% and 15%. In a more specific embodiment, the final concentration ofPEG 4000 is 12±0.5% in the Factor H precipitation step.

In certain embodiments, the methods described above further comprises anenrichment step comprising performing anion exchange chromatography withan enriched Factor H composition. In a specific embodiment, the methodcomprises the steps of (a) contacting a suspended plasma precipitatecomposition containing Factor H and at least one serine protease orserine protease zymogen with finely divided silicon dioxide (SiO₂) underconditions suitable to bind the Factor H, (b) washing the SiO₂ with asolution comprising a pH between 5.0 and 7.0 and a conductivity of lessthan 4 mS/cm, (c) eluting the Factor H from the SiO₂ with a solutioncomprising a pH between 7.0 and 8.0 and a conductivity greater than 10mS/cm, (d) precipitating at least one impurity from the Factor Helution, to form a supernatant comprising Factor H, (e) precipitatingFactor H from the supernatant, (f) re-suspending the precipitatecomprising Factor H, (g) binding Factor H present in the re-suspendedprecipitate to an anion exchange resin, and (h) eluting Factor H fromthe anion exchange resin, thereby providing an enriched Factor Hcomposition. In a preferred embodiment, the serine protease or serineprotease zymogen is one or more of FXI, FXIa, FXII, and FXIIa. Incertain embodiments, the plasma precipitate is a Cohn fraction Iprecipitate, a Cohn fraction II+III precipitate, a Cohn fractionI+II+III precipitate, a Kistler/Nitschmann Precipitate A, aKistler/Nitschmann Precipitate B, or an equivalent fraction thereof. Inone embodiment, the solution used to wash the SiO₂ comprises a pHbetween 5.5 and 6.5. In a specific embodiment, the solution used to washthe SiO₂ comprises a pH of 6.0±0.2. In one embodiment, the solution usedto elute Factor H comprises a conductivity of at least 20 mS/cm. In aspecific embodiment, the solution used to elute Factor H comprises aconductivity of between 25 mS/cm and 40 mS/cm. In one embodiment, theimpurity precipitation step is PEG precipitation. In a specificembodiment, the impurity PEG precipitation comprises precipitation withPEG 4000 at a final concentration between 3% and 7%. In a more specificembodiment, the final concentration of PEG 4000 in the impurityprecipitation step is 5±0.5%. In one embodiment, the Factor Hprecipitation step is PEG precipitation. In a specific embodiment, theFactor H PEG precipitation comprises precipitation with PEG 4000 at afinal concentration between 10% and 15%. In a more specific embodiment,the final concentration of PEG 4000 is 12±0.5% in the Factor Hprecipitation step.

In certain embodiments, the methods described above further comprises anenrichment step comprising performing heparin affinity chromatographywith an enriched Factor H composition. In a specific embodiment, themethod comprises the steps of (a) contacting a suspended plasmaprecipitate composition containing Factor H and at least one serineprotease or serine protease zymogen with finely divided silicon dioxide(SiO₂) under conditions suitable to bind the Factor H, (b) washing theSiO₂ with a solution comprising a pH between 5.0 and 7.0 and aconductivity of less than 4 mS/cm, (c) eluting the Factor H from theSiO₂ with a solution comprising a pH between 7.0 and 8.0 and aconductivity greater than 10 mS/cm, (d) precipitating at least oneimpurity from the Factor H elution, to form a supernatant comprisingFactor H, (e) precipitating Factor H from the supernatant, (f)re-suspending the precipitate comprising Factor H, (g) binding Factor Hpresent in the re-suspended precipitate to an anion exchange resin, (h)eluting Factor H from the anion exchange resin, (i) binding Factor Hpresent in the anion exchange eluate to a heparin affinity resin, and(j) eluting Factor H from the heparin affinity resin, thereby providingan enriched Factor H composition. In a preferred embodiment, the serineprotease or serine protease zymogen is one or more of FXI, FXIa, FXII,and FXIIa. In certain embodiments, the plasma precipitate is a Cohnfraction I precipitate, a Cohn fraction II+III precipitate, a Cohnfraction I+II+III precipitate, a Kistler/Nitschmann Precipitate A, aKistler/Nitschmann Precipitate B, or an equivalent fraction thereof. Inone embodiment, the solution used to wash the SiO₂ comprises a pHbetween 5.5 and 6.5. In a specific embodiment, the solution used to washthe SiO₂ comprises a pH of 6.0±0.2. In one embodiment, the solution usedto elute Factor H comprises a conductivity of at least 20 mS/cm. In aspecific embodiment, the solution used to elute Factor H comprises aconductivity of between 25 mS/cm and 40 mS/cm. In one embodiment, theimpurity precipitation step is PEG precipitation. In a specificembodiment, the impurity PEG precipitation comprises precipitation withPEG 4000 at a final concentration between 3% and 7%. In a more specificembodiment, the final concentration of PEG 4000 in the impurityprecipitation step is 5±0.5%. In one embodiment, the Factor Hprecipitation step is PEG precipitation. In a specific embodiment, theFactor H PEG precipitation comprises precipitation with PEG 4000 at afinal concentration between 10% and 15%. In a more specific embodiment,the final concentration of PEG 4000 is 12±0.5% in the Factor Hprecipitation step.

In certain embodiments, the methods described above further comprisessubjecting a Factor H composition to a dedicated viral removal and/orinactivation step. In a specific embodiment, the method comprises thesteps of (a) contacting a suspended plasma precipitate compositioncontaining Factor H and at least one serine protease or serine proteasezymogen with finely divided silicon dioxide (SiO₂) under conditionssuitable to bind the Factor H, (b) washing the SiO₂ with a solutioncomprising a pH between 5.0 and 7.0 and a conductivity of less than 4mS/cm, (c) eluting the Factor H from the SiO₂ with a solution comprisinga pH between 7.0 and 8.0 and a conductivity greater than 10 mS/cm, (d)precipitating at least one impurity from the Factor H elution, to form asupernatant comprising Factor H, (e) precipitating Factor H from thesupernatant, (f) re-suspending the precipitate comprising Factor H, (g)binding Factor H present in the re-suspended precipitate to an anionexchange resin, (h) eluting Factor H from the anion exchange resin, (i)binding Factor H present in the anion exchange eluate to a heparinaffinity resin, (j) eluting Factor H from the heparin affinity resin,and (k) performing a dedicated viral removal and/or inactivation stepselected from nanofiltration, solvent/detergent (S/D) treatment, heattreatment, and incubation at low pH, thereby providing an enrichedFactor H composition. In a preferred embodiment, the serine protease orserine protease zymogen is one or more of FXI, FXIa, FXII, and FXIIa. Incertain embodiments, the plasma precipitate is a Cohn fraction Iprecipitate, a Cohn fraction II+III precipitate, a Cohn fractionI+II+III precipitate, a Kistler/Nitschmann Precipitate A, aKistler/Nitschmann Precipitate B, or an equivalent fraction thereof. Inone embodiment, the solution used to wash the SiO₂ comprises a pHbetween 5.5 and 6.5. In a specific embodiment, the solution used to washthe SiO₂ comprises a pH of 6.0±0.2. In one embodiment, the solution usedto elute Factor H comprises a conductivity of at least 20 mS/cm. In aspecific embodiment, the solution used to elute Factor H comprises aconductivity of between 25 mS/cm and 40 mS/cm. In one embodiment, theimpurity precipitation step is PEG precipitation. In a specificembodiment, the impurity PEG precipitation comprises precipitation withPEG 4000 at a final concentration between 3% and 7%. In a more specificembodiment, the final concentration of PEG 4000 in the impurityprecipitation step is 5±0.5%. In one embodiment, the Factor Hprecipitation step is PEG precipitation. In a specific embodiment, theFactor H PEG precipitation comprises precipitation with PEG 4000 at afinal concentration between 10% and 15%. In a more specific embodiment,the final concentration of PEG 4000 is 12±0.5% in the Factor Hprecipitation step.

In certain embodiments, the methods described above further comprises astep of concentrating an enriched Factor H composition byultrafiltration/diafiltration. In a specific embodiment, the methodcomprises the steps of (a) contacting a suspended plasma precipitatecomposition containing Factor H and at least one serine protease orserine protease zymogen with finely divided silicon dioxide (SiO₂) underconditions suitable to bind the Factor H, (b) washing the SiO₂ with asolution comprising a pH between 5.0 and 7.0 and a conductivity of lessthan 4 mS/cm, (c) eluting the Factor H from the SiO₂ with a solutioncomprising a pH between 7.0 and 8.0 and a conductivity greater than 10mS/cm, (d) precipitating at least one impurity from the Factor Helution, to form a supernatant comprising Factor H, (e) precipitatingFactor H from the supernatant, (f) re-suspending the precipitatecomprising Factor H, (g) binding Factor H present in the re-suspendedprecipitate to an anion exchange resin, (h) eluting Factor H from theanion exchange resin, (i) binding Factor H present in the anion exchangeeluate to a heparin affinity resin, (j) eluting Factor H from theheparin affinity resin, (k) performing a dedicated viral removal and/orinactivation step selected from nanofiltration, solvent/detergent (S/D)treatment, heat treatment, and incubation at low pH, and (l)concentrating Factor H by ultrafiltration/diafiltration, therebyproviding an enriched Factor H composition. In a preferred embodiment,the serine protease or serine protease zymogen is one or more of FXI,FXIa, FXII, and FXIIa. In certain embodiments, the plasma precipitate isa Cohn fraction I precipitate, a Cohn fraction II+III precipitate, aCohn fraction I+II+III precipitate, a Kistler/Nitschmann Precipitate A,a Kistler/Nitschmann Precipitate B, or an equivalent fraction thereof.In one embodiment, the solution used to wash the SiO₂ comprises a pHbetween 5.5 and 6.5. In a specific embodiment, the solution used to washthe SiO₂ comprises a pH of 6.0±0.2. In one embodiment, the solution usedto elute Factor H comprises a conductivity of at least 20 mS/cm. In aspecific embodiment, the solution used to elute Factor H comprises aconductivity of between 25 mS/cm and 40 mS/cm. In one embodiment, theimpurity precipitation step is PEG precipitation. In a specificembodiment, the impurity PEG precipitation comprises precipitation withPEG 4000 at a final concentration between 3% and 7%. In a more specificembodiment, the final concentration of PEG 4000 in the impurityprecipitation step is 5±0.5%. In one embodiment, the Factor Hprecipitation step is PEG precipitation. In a specific embodiment, theFactor H PEG precipitation comprises precipitation with PEG 4000 at afinal concentration between 10% and 15%. In a more specific embodiment,the final concentration of PEG 4000 is 12±0.5% in the Factor Hprecipitation step.

C. Inter-alpha-Trypsin Inhibitor (IαI)

In one embodiment, the present invention provides a method for reducingthe amount of a serine protease or a serine protease zymogen in aplasma-derived IαI composition. In one specific embodiment, the methodcomprises the steps of: (a) contacting the IαI composition with finelydivided silicon dioxide (SiO₂) under conditions suitable to bind atleast one serine protease or serine protease zymogen; and (b) separatingthe SiO₂ from the IαI composition to remove the bound serine protease orserine protease zymogen. In a preferred embodiment, the serine proteaseor serine protease zymogen is Factor XIa (FXIa), Factor XIIa (FXIIa),Factor XI (FXI), and/or Factor XII (FXII).

In one embodiment, the method further comprises the step of performing afirst IαI protein enrichment step to form a first enriched IαIcomposition, prior to contacting the composition with finely dividedsilicon dioxide (SiO₂). In certain embodiments, the first IαI proteinenrichment step is selected from a protein precipitation step (e.g., analcohol fractionation step), an ultrafiltration/diafiltration step, anda chromatographic step.

In certain embodiments, the methods described above further comprisesthe step of performing a second IαI protein enrichment step to form asecond enriched IαI composition, prior to contacting the compositionwith finely divided silicon dioxide (SiO₂). In certain embodiments, thefirst IαI protein enrichment step is selected from a proteinprecipitation step (e.g., an alcohol fractionation step), anultrafiltration/diafiltration step, and a chromatographic step.

Accordingly, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived IαI composition, the method comprises the steps of: (a)performing a first IαI enrichment step to form a first enrichedplasma-derived IαI composition; (b) performing a second IαI enrichmentstep to form a second enriched plasma-derived IαI composition; (c)contacting the second enriched composition with finely divided silicondioxide (SiO₂) under conditions suitable to bind at least one serineprotease or serine protease zymogen; and (d) separating the SiO₂ fromthe composition to remove the bound serine protease or serine proteasezymogen. In a preferred embodiment, the serine protease or serineprotease zymogen is Factor XIa (FXIa), Factor XIIa (FXIIa), Factor XI(FXI), and/or Factor XII (FXII). In certain embodiments, the combinationof first and second enrichment steps is selected from any one ofvariations Var. 1 to Var. 100, found in Table 1.

In certain embodiments, the methods described above further comprisesthe step of performing an IαI enrichment step after contacting thecomposition with finely divided silicon dioxide (SiO₂). In certainembodiments, the IαI enrichment step is selected from a proteinprecipitation step (e.g., an alcohol fractionation step), anultrafiltration/diafiltration step, and a chromatographic step.

Accordingly, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived IαI composition the method comprises the steps of: (a)performing a first IαI enrichment step to form a first enrichedplasma-derived IαI composition; (b) contacting the first enrichedcomposition with finely divided silicon dioxide (SiO₂) under conditionssuitable to bind at least one serine protease or serine proteasezymogen; (c) separating the SiO₂ from the composition to remove thebound serine protease or serine protease zymogen; and (d) performing asecond IαI enrichment step to form a second enriched plasma-derived IαIcomposition. In a preferred embodiment, the serine protease or serineprotease zymogen is Factor XIa (FXIa), Factor XIIa (FXIIa), Factor XI(FXI), and/or Factor XII (FXII). In certain embodiments, the combinationof first and second enrichment steps is selected from any one ofvariations Var. 1 to Var. 100, found in Table 1.

Likewise, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived IαI composition, the method comprising the steps of:(a) performing a first IαI enrichment step to form a first enrichedplasma-derived IαI composition; (b) performing a second IαI enrichmentstep to form a second enriched plasma-derived IαI composition; (c)contacting the second enriched composition with finely divided silicondioxide (SiO₂) under conditions suitable to bind at least one serineprotease or serine protease zymogen; (d) separating the SiO₂ from thecomposition to remove the bound serine protease or serine proteasezymogen; and (e) performing a third IαI enrichment step to form a thirdenriched plasma-derived IαI composition. In a preferred embodiment, theserine protease or serine protease zymogen is Factor XIa (FXIa), FactorXIIa (FXIIa), Factor XI (FXI), and/or Factor XII (FXII). In certainembodiments, the combination of first and second enrichment steps isselected from any one of variations Var. 101 to Var. 1100, found inTable 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9,Table 10, or Table 11.

1. Co-Binding and Differential Elution

In one aspect, the present invention provides a method for preparing aplasma-derived IαI composition having a reduced amount of a serineprotease or a serine protease zymogen, the method comprisingco-extracting IαI and a serine protease and/or serine protease zymogenfrom a composition derived from pooled plasma by binding the proteins tofinely divided silicon dioxide (SiO₂), eluting the serine proteaseand/or serine protease zymogen from the SiO₂ under a first solutioncondition, and subsequently eluting IαI from the SiO₂ under a secondsolution condition. In a preferred embodiment, the starting compositionis a re-suspended Fraction II+III precipitate or equivalent precipitatethereof.

In a specific embodiment, the method comprises the steps of: (a)contacting a composition containing IαI and at least one serine proteaseor serine protease zymogen with finely divided silicon dioxide (SiO₂)under conditions suitable to bind the IαI and at least one serineprotease or serine protease zymogen; (b) separating the SiO₂ from thecomposition; (c) eluting the serine protease or serine protease zymogenfrom the SiO₂ under a solution condition in which the IαI remains bound;and (d) eluting the IαI from the SiO₂.

In certain embodiments, a solution condition in which the IαI remainsbound refers to a condition that preferentially elutes the serineprotease or serine protease zymogen, while a substantial fraction of IαIremains bound to the SiO₂. In one embodiment, a substantial fractionrefers to at least 10% of the IαI bound to the SiO₂. In anotherembodiment, a substantial fraction refers to at least 25% of the IαIbound to the SiO₂. In another embodiment, a substantial fraction refersto at least 50% of the IαI bound to the SiO₂. In another embodiment, asubstantial fraction refers to at least 75% of the IαI bound to theSiO₂. In yet other embodiments, a substantial fraction refers to atleast 10% of the IαI bound to the SiO₂, or at least 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or moreof the IαI bound to the SiO₂.

In certain embodiments, differential elution of the serine protease orserine protease zymogen and IαI is achieved by sequentially contacting(i.e., step-wise elution) the SiO₂ with a first solution condition(e.g., a first elution buffer) suitable to elute the majority of theserine protease or serine protease zymogen but not a substantialfraction of the bound IαI, and a second solution condition (e.g., asecond elution buffer) suitable to elute the substantial fraction ofbound IαI from the SiO₂.

In other embodiments, differential elution of the serine protease orserine protease zymogen and IαI is achieved by gradually changing thesolution conditions (i.e., with an elution gradient) from a firstsolution condition suitable to elute the majority of the serine proteaseor serine protease zymogen but not a substantial fraction of the boundIαI to a second solution condition suitable to elute the substantialfraction of bound IαI from the SiO₂. In this fashion, the serineprotease or serine protease zymogen and IαI content eluted off of theSiO₂ may be partially overlapping. By fractionating the elution andcharacterizing the individual fractions, a IαI pool may be created fromfractions having high IαI content and low serine protease or serineprotease zymogen content.

2. Co-Binding and Preferential IαI Elution

In one aspect, the present invention provides a method for preparing aplasma-derived IαI composition having a reduced amount of a serineprotease or a serine protease zymogen, the method comprisingco-extracting IαI and a serine protease and/or serine protease zymogenfrom a composition derived from pooled plasma by binding the proteins tofinely divided silicon dioxide (SiO₂), and eluting the IαI from the SiO₂under conditions in which a substantial fraction of the bound serineprotease and/or serine protease zymogen remains bound to the SiO₂. In apreferred embodiment, the starting composition is a re-suspendedFraction II+III precipitate or equivalent precipitate thereof.

In a specific embodiment, the method comprises the steps of: (a)contacting a composition containing IαI and at least one serine proteaseor serine protease zymogen with finely divided silicon dioxide (SiO₂)under conditions suitable to bind the IαI and at least one serineprotease or serine protease zymogen; (b) separating the SiO₂ from thecomposition; and (c) eluting the IαI from the SiO₂ under a solutioncondition in which the serine protease or serine protease zymogenremains bound.

In certain embodiments, a solution condition in which the serineprotease or serine protease zymogen remains bound refers to a conditionthat preferentially elutes the IαI, while a substantial fraction of theserine protease or serine protease zymogen remains bound to the SiO₂. Inone embodiment, a substantial fraction refers to at least 10% of theserine protease or serine protease zymogen bound to the SiO₂. In anotherembodiment, a substantial fraction refers to at least 25% of the serineprotease or serine protease zymogen bound to the SiO₂. In anotherembodiment, a substantial fraction refers to at least 50% of the serineprotease or serine protease zymogen bound to the SiO₂. In anotherembodiment, a substantial fraction refers to at least 75% of the serineprotease or serine protease zymogen bound to the SiO₂. In yet otherembodiments, a substantial fraction refers to at least 10% of the serineprotease or serine protease zymogen bound to the SiO₂, or at least 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97,98, 99, or more of the serine protease or serine protease zymogen boundto the SiO₂.

3. Preferential Binding of IαI

In one aspect, the present invention provides a method for preparing aplasma-derived IαI composition having a reduced amount of a serineprotease or a serine protease zymogen, the method comprising (a)contacting a composition containing IαI and at least one serine proteaseor serine protease zymogen with finely divided silicon dioxide (SiO₂)under conditions suitable to bind the IαI but not the at least oneserine protease or serine protease zymogen; (b) separating the SiO₂ fromthe composition; and (c) eluting the IαI from the SiO₂.

In certain embodiments, a solution condition in which the serineprotease or serine protease zymogen does not bind to the SiO₂ refers toa condition that preferentially allows IαI binding to the SiO₂, while asubstantial fraction of the serine protease or serine protease zymogenremains unbound in the solution. In one embodiment, a substantialfraction refers to at least 10% of the serine protease or serineprotease zymogen in the starting composition. In another embodiment, asubstantial fraction refers to at least 25% of the serine protease orserine protease zymogen in the starting composition. In anotherembodiment, a substantial fraction refers to at least 50% of the serineprotease or serine protease zymogen in the starting composition. Inanother embodiment, a substantial fraction refers to at least 75% of theserine protease or serine protease zymogen in the starting composition.In yet other embodiments, a substantial fraction refers to at least 10%of the serine protease or serine protease zymogen in the startingcomposition, or at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 96, 97, 98, 99, or more of the serine protease orserine protease zymogen in the starting composition.

4. Preferential Binding of Serine Protease or Serine Protease Zymogen

In one aspect, the present invention provides a method for preparing aplasma-derived IαI composition having a reduced amount of a serineprotease or a serine protease zymogen, the method comprising (a)contacting a composition containing IαI and at least one serine proteaseor serine protease zymogen with finely divided silicon dioxide (SiO₂)under conditions suitable to bind the serine protease and/or serineprotease zymogen but not the IαI; and (b) separating the SiO₂ from thecomposition.

In certain embodiments, a solution condition in which the IαI does notbind to the SiO₂ refers to a condition that preferentially allows serineprotease or serine protease zymogen binding to the SiO₂, while asubstantial fraction of the IαI remains unbound in the solution. In oneembodiment, a substantial fraction refers to at least 10% of the IαI inthe starting composition. In another embodiment, a substantial fractionrefers to at least 25% of the IαI in the starting composition. Inanother embodiment, a substantial fraction refers to at least 50% of theIαI in the starting composition. In another embodiment, a substantialfraction refers to at least 75% of the IαI in the starting composition.In yet other embodiments, a substantial fraction refers to at least 10%of the IαI in the starting composition, or at least 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or moreof the IαI in the starting composition.

D. Alpha-1-Antitrypsin (A1PI)

In one embodiment, the present invention provides a method for reducingthe amount of a serine protease or a serine protease zymogen in aplasma-derived alpha-1-antitrypsin (A1PI) composition. In one specificembodiment, the method comprises the steps of: (a) contacting an A1PIcomposition with finely divided silicon dioxide (SiO₂) under conditionssuitable to bind at least one serine protease or serine proteasezymogen; and (b) separating the SiO₂ from the A1PI composition to removethe bound serine protease or serine protease zymogen. In a preferredembodiment, the serine protease or serine protease zymogen is Factor XIa(FXIa), Factor XIIa (FXIIa), Factor XI (FXI), and/or Factor XII (FXII).

In one embodiment, the method further comprises the step of performing afirst A1PI protein enrichment step to form a first enriched A1PIcomposition, prior to contacting the composition with finely dividedsilicon dioxide (SiO₂). In certain embodiments, the first A1PI proteinenrichment step is selected from a protein precipitation step (e.g., analcohol fractionation step), an ultrafiltration/diafiltration step, anda chromatographic step.

In certain embodiments, the methods described above further comprisesthe step of performing a second A1PI protein enrichment step to form asecond enriched A1PI composition, prior to contacting the compositionwith finely divided silicon dioxide (SiO₂). In certain embodiments, thefirst A1PI protein enrichment step is selected from a proteinprecipitation step (e.g., an alcohol fractionation step), anultrafiltration/diafiltration step, and a chromatographic step.

Accordingly, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived A1PI composition, the method comprises the steps of:(a) performing a first A1PI enrichment step to form a first enrichedplasma-derived A1PI composition; (b) performing a second A1PI enrichmentstep to form a second enriched plasma-derived A1PI composition; (c)contacting the second enriched composition with finely divided silicondioxide (SiO₂) under conditions suitable to bind at least one serineprotease or serine protease zymogen; and (d) separating the SiO₂ fromthe composition to remove the bound serine protease or serine proteasezymogen. In a preferred embodiment, the serine protease or serineprotease zymogen is Factor XIa (FXIa), Factor XIIa (FXIIa), Factor XI(FXI), and/or Factor XII (FXII). In certain embodiments, the combinationof first and second enrichment steps is selected from any one ofvariations Var. 1 to Var. 100, found in Table 1.

In certain embodiments, the methods described above further comprisesthe step of performing an A1PI enrichment step after contacting thecomposition with finely divided silicon dioxide (SiO₂). In certainembodiments, the A1PI enrichment step is selected from a proteinprecipitation step (e.g., an alcohol fractionation step), anultrafiltration/diafiltration step, and a chromatographic step.

Accordingly, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived A1PI composition the method comprises the steps of: (a)performing a first A1PI enrichment step to form a first enrichedplasma-derived A1PI composition; (b) contacting the first enrichedcomposition with finely divided silicon dioxide (SiO₂) under conditionssuitable to bind at least one serine protease or serine proteasezymogen; (c) separating the SiO₂ from the composition to remove thebound serine protease or serine protease zymogen; and (d) performing asecond A1PI enrichment step to form a second enriched plasma-derivedA1PI composition. In a preferred embodiment, the serine protease orserine protease zymogen is Factor XIa (FXIa), Factor XIIa (FXIIa),Factor XI (FXI), and/or Factor XII (FXII). In certain embodiments, thecombination of first and second enrichment steps is selected from anyone of variations Var. 1 to Var. 100, found in Table 1.

Likewise, in one embodiment, the invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived A1PI composition, the method comprising the steps of:(a) performing a first A1PI enrichment step to form a first enrichedplasma-derived A1PI composition; (b) performing a second A1PI enrichmentstep to form a second enriched plasma-derived Ig composition; (c)contacting the second enriched composition with finely divided silicondioxide (SiO₂) under conditions suitable to bind at least one serineprotease or serine protease zymogen; (d) separating the SiO₂ from thecomposition to remove the bound serine protease or serine proteasezymogen; and (e) performing a third A1PI enrichment step to form a thirdenriched plasma-derived A1PI composition. In a preferred embodiment, theserine protease or serine protease zymogen is Factor XIa (FXIa), FactorXIIa (FXIIa), Factor XI (FXI), and/or Factor XII (FXII). In certainembodiments, the combination of first and second enrichment steps isselected from any one of variations Var. 101 to Var. 1100, found inTable 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9,Table 10, or Table 11.

In a particular embodiment, the A1PI composition is a manufacturingintermediate. For example, in certain embodiments, the A1PI compositionis a manufacturing intermediate from a Cohn fractionation procedure (J.Am. Chem. Soc., 1946, 68(3): 459-475; J. Am. Chem. Soc. 72:465-474(1950)), an Oncley fractionation procedure (J. Am. Chem. Soc., 1949,71(2): 541-550), a Kistler/Nitschmann fractionation procedure (Vox Sang.7:414-424 (1962)), a purification procedure disclosed in U.S. Pat. No.6,974,792 or 7,807,435, modified procedures thereof, and similar orequivalent purification procedures known in the art. The aforementionedreferences are hereby incorporated by reference in their entireties forall purposes.

For example, a number of production methods for A1PI are known whichcomprise the fractionated precipitation of plasma with polyethyleneglycol 4000, but also the processing of various plasma fractions (Cohnfraction IV-1-precipitate or Kistler and Nitschmann Supernatant A orA+1) (Feldman and Winkelman, Blood Separation and Plasma Fractionation(1991), Wiley-Liss, Inc., pp. 341-383). In more elaborate purifications,the respective blood fractions have been purified by means of DEAEcellulose, e.g. (Basis et al. (Vopr. Med. Khim. 33 (1) (1987), 54-59)),treated with affinity chromatographic materials or with cation exchangerchromatographic materials (EP 0 698 615 A1). U.S. Pat. No. 6,974,792describes a purification process yielding A1PI with high specificactivity utilizing a Cohn fraction V precipitate. U.S. Pat. No.7,807,435 describes a purification process providing higher yields ofA1PI, utilizing a Cohn fraction IV-1 and/or fraction IV-4 precipitate.

In one particular embodiment, the A1PI composition is a cryo-poor Cohnpool. In another particular embodiment, the A1PI composition is are-suspended Cohn Fraction V precipitate or equivalent fraction thereof.In another particular embodiment, the A1PI composition is a re-suspendedCohn Fraction IV-1 precipitate, or equivalent fraction thereof. Inanother particular embodiment, the A1PI composition is a re-suspendedCohn Fraction IV-4 precipitate, or equivalent fraction thereof. Inanother particular embodiment, the A1PI composition is aKistler/Nitschmann Supernatant A, or equivalent fraction thereof.

Generally, serine protease and/or serine protease zymogen removal fromA1PI compositions can be achieved by treating the A1PI-containingcomposition with finely divided silicon dioxide (SiO₂) under pH andconductivity solution conditions in which the serine protease and/orserine protease zymogen binds to the SiO₂.

In one embodiment, the process improvements are realized by inclusion ofa fumed silica treatment prior to filtration or centrifugalclarification of a plasma precipitate comprising A1PI. In oneembodiment, the SiO₂ treatment step comprises addition of finely dividedsilica dioxide particles (e.g., fumed silica, Aerosil®) followed by a 40minute to 16 hour incubation period during which the suspension isconstantly mixed. In certain embodiments, the incubation period will bebetween about 50 minutes and about 70 minutes, or about 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, or more minutes. In otherembodiments, the incubation period will be at least 1 hour, or at least2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours,10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, ormore hours. In a particular embodiment, the incubation period will be atleast 15 hours. Generally, the treatment will be performed at betweenabout 0° C. and about 25° C., or between about 2° C. and about 8° C. Incertain embodiments, the treatment may be performed at about 0° C., 1°C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11°C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., 20°C., 21° C., 22° C., 23° C., 24° C., or 25° C. In a particularembodiment, the treatment is performed at between about 2° C. and about25° C. In a specific embodiment, the process improvements are realizedby inclusion of a fumed silica treatment, which reduces the levels ofFXI, FXIa, FXII, and FXIIa in the immunoglobulin preparation.

In certain embodiments, fumed silica is added at a concentration ofbetween about 20 g/kg precipitate and about 100 g/kg precipitate. Incertain embodiments, the fumed silica may be added at a concentration ofabout 20 g/kg precipitate, or about 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 g/kg precipitate. In one specificembodiment, fumed silica (e.g., Aerosil 380 or equivalent) is added tothe precipitate re-suspension to a final concentration of about 40 g/kgprecipitate.

In certain embodiments, SiO₂ is added to an A1PI composition at aconcentration between about 0.01 g/g protein and about 10 g/g protein.In another embodiment, SiO₂ is added to an A1PI composition at aconcentration between about 0.01 g/g protein and about 5 g/g protein. Inanother embodiment, SiO₂ is added to an A1PI composition at aconcentration between about 0.02 g/g protein and about 4 g/g protein. Inone embodiment, SiO₂ is added to an A1PI composition at a finalconcentration of at least 0.1 g per gram total protein. In anotherspecific embodiment, fumed silica is added at a concentration of atleast 0.2 g per gram total protein. In another specific embodiment,fumed silica is added at a concentration of at least 0.25 g per gramtotal protein. In other specific embodiments, fumed silica is added at aconcentration of at least 1 g per gram total protein. In anotherspecific embodiment, fumed silica is added at a concentration of atleast 2 g per gram total protein. In another specific embodiment, fumedsilica is added at a concentration of at least 2.5 g per gram totalprotein. In yet other specific embodiments, finely divided silicondioxide is added at a concentration of at least 0.01 g/g total proteinor at least 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, or more g/gtotal protein.

In certain embodiments, filter aid, for example Celpure C300 (Celpure)or Hyflo-Supper-Cel (World Minerals), will be added after the silicadioxide treatment, to facilitate depth filtration. Filter aid can beadded at a final concentration of from about 0.01 kg/kg precipitate toabout 1.0 kg/kg precipitate, or from about 0.02 kg/kg precipitate toabout 0.8 kg/kg precipitate, or from about 0.03 kg/kg precipitate toabout 0.7 kg/kg precipitate. In certain embodiments, the filter aid willbe added at a final concentration of at least 0.01 kg/kg precipitate, orat least 0.02 kg/kg, 0.03 kg/kg, 0.04 kg/kg, 0.05 kg/kg, 0.06 kg/kg,0.07 kg/kg, 0.08 kg/kg, 0.09 kg/kg, 0.1 kg/kg, 0.2 kg/kg, 0.3 kg/kg, 0.4kg/kg, 0.5 kg/kg, 0.6 kg/kg, 0.7 kg/kg, 0.8 kg/kg, 0.9 kg/kg, or 1.0kg/kg precipitate. In certain embodiments, the filter aid will be addedat a final concentration of about 0.01 kg/kg precipitate, or about 0.02kg/kg, 0.03 kg/kg, 0.04 kg/kg, 0.05 kg/kg, 0.06 kg/kg, 0.07 kg/kg, 0.08kg/kg, 0.09 kg/kg, 0.1 kg/kg, 0.2 kg/kg, 0.3 kg/kg, 0.4 kg/kg, 0.5kg/kg, 0.6 kg/kg, 0.7 kg/kg, 0.8 kg/kg, 0.9 kg/kg, or 1.0 kg/kgprecipitate.

Accordingly, in one embodiment, the present invention provides a methodfor reducing the amount of a serine protease or a serine proteasezymogen in a plasma-derived A1PI composition, the method comprisingcontacting the composition with SiO₂ at a pH between about 4.0 and about7.0 to bind a serine protease or a serine protease zymogen. In anotherembodiment, the method comprises contacting the composition with SiO₂ ata pH between about 4.0 and about 6.5. In another embodiment, the methodcomprises contacting the composition with SiO₂ at a pH between about 4.0and about 6.0. In another embodiment, the method comprises contactingthe composition with SiO₂ at a pH between about 4.0 and about 5.5. Inanother embodiment, the method comprises contacting the composition withSiO₂ at a pH between about 4.0 and about 5.0. In another embodiment, themethod comprises contacting the composition with SiO₂ at a pH betweenabout 4.5 and about 7.0. In another embodiment, the method comprisescontacting the composition with SiO₂ at a pH between about 4.5 and about6.5. In another embodiment, the method comprises contacting thecomposition with SiO₂ at a pH between about 4.5 and about 6.0. Inanother embodiment, the method comprises contacting the composition withSiO₂ at a pH between about 4.5 and about 5.5. In another embodiment, themethod comprises contacting the composition with SiO₂ at a pH betweenabout 4.5 and about 5.0. In another embodiment, the method comprisescontacting the composition with SiO₂ at a pH between about 5.0 and about7.0. In another embodiment, the method comprises contacting thecomposition with SiO₂ at a pH between about 5.0 and about 6.5. Inanother embodiment, the method comprises contacting the composition withSiO₂ at a pH between about 5.0 and about 6.0. In another embodiment, themethod comprises contacting the composition with SiO₂ at a pH betweenabout 5.0 and about 5.5. In yet another embodiment, the method comprisescontacting the composition with SiO₂ at a pH between about 4.6 and about5.6. In another embodiment, the method comprises contacting thecomposition with SiO₂ at a pH between about 4.7 and about 5.5. Inanother embodiment, the method comprises contacting the composition withSiO₂ at a pH between about 4.8 and about 5.4. In another embodiment, themethod comprises contacting the composition with SiO₂ at a pH betweenabout 4.9 and about 5.3. In another embodiment, the method comprisescontacting the composition with SiO₂ at a pH between about 5.0 and about5.2. In another embodiment, the method comprises contacting thecomposition with SiO₂ at a pH of about 5.1. In other embodiments, themethod comprises contacting the composition with SiO₂ at a pH of about4.0 or about 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, or no more than 7.0. In yet other embodiments, the methodcomprises contacting the composition with SiO₂ at a pH of no more than4.0 or no more than 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, or no more than 7.0.

In one embodiment, the present invention provides a method for reducingthe amount of a serine protease or a serine protease zymogen in aplasma-derived A1PI composition, the method comprising contacting thecomposition with SiO₂ at an ionic strength between about 0.1 mS/cm andabout 2.0 mS/cm to bind a serine protease or a serine protease zymogen.In another embodiment, the method comprises contacting the compositionwith SiO₂ at an ionic strength between about 0.1 mS/cm and about 1.9mS/cm. In another embodiment, the method comprises contacting thecomposition with SiO₂ at an ionic strength between about 0.1 mS/cm andabout 1.8 mS/cm. In another embodiment, the method comprises contactingthe composition with SiO₂ at an ionic strength between about 0.1 mS/cmand about 1.7 mS/cm. In another embodiment, the method comprisescontacting the composition with SiO₂ at an ionic strength between about0.1 mS/cm and about 1.6 mS/cm. In another embodiment, the methodcomprises contacting the composition with SiO₂ at an ionic strengthbetween about 0.1 mS/cm and about 1.5 mS/cm. In another embodiment, themethod comprises contacting the composition with SiO₂ at an ionicstrength between about 0.1 mS/cm and about 1.4 mS/cm. In anotherembodiment, the method comprises contacting the composition with SiO₂ atan ionic strength between about 0.1 mS/cm and about 1.3 mS/cm. Inanother embodiment, the method comprises contacting the composition withSiO₂ at an ionic strength between about 0.1 mS/cm and about 1.2 mS/cm.In another embodiment, the method comprises contacting the compositionwith SiO₂ at an ionic strength between about 0.1 mS/cm and about 1.1mS/cm. In another embodiment, the method comprises contacting thecomposition with SiO₂ at an ionic strength between about 0.1 mS/cm andabout 1.0 mS/cm. In another embodiment, the method comprises contactingthe composition with SiO₂ at an ionic strength between about 0.1 mS/cmand about 0.9 mS/cm. In another embodiment, the method comprisescontacting the composition with SiO₂ at an ionic strength between about0.1 mS/cm and about 0.8 mS/cm. In another embodiment, the methodcomprises contacting the composition with SiO₂ at an ionic strengthbetween about 0.2 mS/cm and about 1.0 mS/cm. In another embodiment, themethod comprises contacting the composition with SiO₂ at an ionicstrength between about 0.3 mS/cm and about 1.0 mS/cm. In anotherembodiment, the method comprises contacting the composition with SiO₂ atan ionic strength between about 0.1 mS/cm and about 0.4 mS/cm. Inanother embodiment, the method comprises contacting the composition withSiO₂ at an ionic strength between about 0.5 mS/cm and about 1.0 mS/cm.In another embodiment, the method comprises contacting the compositionwith SiO₂ at an ionic strength between about 0.6 mS/cm and about 1.0mS/cm. In another embodiment, the method comprises contacting thecomposition with SiO₂ at an ionic strength between about 0.7 mS/cm andabout 0.9 mS/cm. In another embodiment, the method comprises contactingthe composition with SiO₂ at an ionic strength of about 0.8 mS/cm. Inother embodiments, the method comprises contacting the composition withSiO₂ at an ionic strength of about 0.1 mS/cm or no more than 0.2 mS/cm,0.3 mS/cm, 0.4 mS/cm, 0.5 mS/cm, 0.6 mS/cm, 0.7 mS/cm, 0.8 mS/cm, 0.9mS/cm, 1.0 mS/cm, 1.1 mS/cm, 1.2 mS/cm, 1.3 mS/cm, 1.4 mS/cm, 1.5 mS/cm,1.6 mS/cm, 1.7 mS/cm, 1.8 mS/cm, 1.9 mS/cm, 2.0 mS/cm, 2.1 mS/cm, 2.2mS/cm, 2.3 mS/cm, 2.4 mS/cm, 2.5 mS/cm, 2.6 mS/cm, 2.7 mS/cm, 2.8 mS/cm,2.9 mS/cm, or 3.0 mS/cm. In yet other embodiments, the method comprisescontacting the composition with SiO₂ at an ionic strength of no morethan 0.1 mS/cm or no more than 0.2 mS/cm, 0.3 mS/cm, 0.4 mS/cm, 0.5mS/cm, 0.6 mS/cm, 0.7 mS/cm, 0.8 mS/cm, 0.9 mS/cm, 1.0 mS/cm, 1.1 mS/cm,1.2 mS/cm, 1.3 mS/cm, 1.4 mS/cm, 1.5 mS/cm, 1.6 mS/cm, 1.7 mS/cm, 1.8mS/cm, 1.9 mS/cm, 2.0 mS/cm, 2.1 mS/cm, 2.2 mS/cm, 2.3 mS/cm, 2.4 mS/cm,2.5 mS/cm, 2.6 mS/cm, 2.7 mS/cm, 2.8 mS/cm, 2.9 mS/cm, or 3.0 mS/cm.

In certain embodiments, the present invention provides a method forreducing the amount of a serine protease or a serine protease zymogen ina plasma-derived A1PI composition, the method comprising contacting thecomposition with SiO₂ at a low pH and low ionic strength to bind aserine protease or a serine protease zymogen. In a particularembodiment, the method comprises contacting the composition with SiO₂ ata pH between about 4.8 and about 5.4 at an ionic strength between about0.6 mS/cm and about 1.0 mS/cm. In a more particular embodiment, themethod comprises contacting the composition with SiO₂ at a pH betweenabout 4.9 and about 5.3 at an ionic strength between about 0.7 mS/cm andabout 0.9 mS/cm. In a yet more particular embodiment, the methodcomprises contacting the composition with SiO₂ at a pH between about 5.0and about 5.2 at an ionic strength of about 0.8 mS/cm. In yet otherembodiments, the method comprises contacting the composition with SiO₂at a pH and ionic strength according to any one of variations Var. 1222to 3041, as presented in Table 12, Table 13, Table 14, and Table 15.

1. Binding and Elution of Serine Proteases or Serine Protease Zymogens

In one aspect, the present invention provides a method for reducing theamount of serine protease and/or serine protease zymogen in are-suspended plasma precipitate comprising A1PI. Generally, theprecipitate may be any precipitated during the fractionation of pooledplasma, preferably human plasma. In one embodiment, the method comprisescontacting a re-suspended plasma precipitate comprising A1PI in aninsoluble state with finely divided silicon dioxide (SiO₂) under a firstlow pH solution condition to bind the serine protease and/or serineprotease zymogen and to maintain the A1PI in an insoluble state,separating the soluble and insoluble portions of the suspension, elutingthe serine and/or serine protease zymogen from SiO₂ under a second lowpH solution condition suitable to maintain a substantial fraction of theA1PI in an insoluble state, separating the soluble and insolubleportions of the suspension, and extracting the A1PI from the insolubleportion. In one embodiment, the SiO₂ is admixed prior to or during theprecipitation reaction and recovered along with the precipitate. In aspecific embodiment, the precipitate is a Cohn fraction IV-1precipitate. In another embodiment, the precipitate is a Cohn fractionIV-4 precipitate. In another embodiment, the precipitate is a Cohnfraction V precipitate. In another embodiment, the precipitate is aKistler/Nitschmann precipitate IV. In yet another embodiment, theprecipitate is a Kistler/Nitschmann precipitate C.

In one embodiment of the methods provided above, the first low pHsolution condition comprises a pH of between 4.0 and 7.0 and an ionicstrength of less than about 5.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 7.0 and an ionicstrength of less than about 5.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 6.5 and an ionicstrength of less than about 5.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 6.0 and an ionicstrength of less than about 5.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.5 and 6.0 and an ionicstrength of less than about 5.0 mS/cm. In a specific embodiment, thesolution condition comprises a pH of 5.5±0.2 and an ionic strength ofless than about 5.0 mS/cm. In another specific embodiment, the solutioncondition comprises a pH of 6.0±0.2 and an ionic strength of less thanabout 5.0 mS/cm.

In another embodiment of the methods provided above, the first low pHsolution condition comprises a pH of between 5.0 and 6.5 and an ionicstrength of less than about 4.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 6.5 and an ionicstrength of less than about 3.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 6.5 and an ionicstrength of less than about 2.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 6.5 and an ionicstrength of less than about 1.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 6.5 and an ionicstrength of less than about 0.5 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.5 and 6.0 and an ionicstrength of less than about 4.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.5 and 6.0 and an ionicstrength of less than about 3.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.5 and 6.0 and an ionicstrength of less than about 2.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.5 and 6.0 and an ionicstrength of less than about 1.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.5 and 6.0 and an ionicstrength of less than about 0.5 mS/cm. In a specific embodiment, thesolution conditions comprises a pH of 5.5±0.2 and an ionic strength ofless than about 3.0 mS/cm. In another specific embodiment, the solutioncondition comprises a pH of 6.0±0.2 and an ionic strength of less thanabout 3.0 mS/cm. In yet other embodiments, the first low pH solutioncondition comprises a pH and ionic strength according to any one ofvariations Var. 1222 to 3041, as presented in Table 12, Table 13, Table14, and Table 15.

In one embodiment of the methods provided above, the second low pHsolution condition comprises a pH of between 4.0 and 7.0 and an ionicstrength of greater than about 5.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 7.0 and an ionicstrength of greater than about 5.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 6.5 and an ionicstrength of greater than about 5.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 6.0 and an ionicstrength of greater than about 5.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.5 and 6.0 and an ionicstrength of greater than about 5.0 mS/cm. In a specific embodiment, thesolution condition comprises a pH of 5.5±0.2 and an ionic strength ofgreater than about 5.0 mS/cm. In another specific embodiment, thesolution conditions comprises a pH of 6.0±0.2 and an ionic strength ofgreater than about 5.0 mS/cm.

In another embodiment of the methods provided above, the second low pHsolution condition comprises a pH of between 5.0 and 6.5 and an ionicstrength of greater than about 3.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 6.5 and an ionicstrength of greater than about 4.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 6.5 and an ionicstrength of greater than about 6.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 6.5 and an ionicstrength of greater than about 7.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.0 and 6.5 and an ionicstrength of greater than about 10 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.5 and 6.0 and an ionicstrength of greater than about 3.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.5 and 6.0 and an ionicstrength of greater than about 4.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.5 and 6.0 and an ionicstrength of greater than about 6.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.5 and 6.0 and an ionicstrength of greater than about 7.0 mS/cm. In another embodiment, thesolution condition comprises a pH of between 5.5 and 6.0 and an ionicstrength of greater than about 10 mS/cm. In a specific embodiment, thesolution conditions comprises a pH of 5.5±0.2 and an ionic strength ofgreater than about 10 mS/cm. In another specific embodiment, thesolution condition comprises a pH of 6.0±0.2 and an ionic strength ofgreater than about 10 mS/cm.

In one specific embodiment, the present invention provides a method forreducing the amount of serine protease and/or serine protease zymogen ina re-suspended plasma precipitate comprising A1PI, comprising the stepsof contacting a re-suspended plasma precipitate comprising A1PI in aninsoluble state with finely divided silicon dioxide (SiO₂) under a firstlow pH solution condition comprising a pH between about 5.0 and about6.5 and an ionic strength of less than 5.0 mS to bind the serineprotease and/or serine protease zymogen and to maintain the A1PI in aninsoluble state, separating the soluble and insoluble portions of thesuspension, eluting the serine and/or serine protease zymogen from SiO₂under a second low pH solution condition comprising a pH between about5.0 and about 6.5 and an ionic strength of greater than 5.0 mS tomaintain a substantial fraction of the A1PI in an insoluble state,separating the soluble and insoluble portions of the suspension, andextracting the A1PI from the insoluble portion. In one embodiment, theSiO₂ is admixed prior to or during the precipitation reaction andrecovered along with the precipitate. In a specific embodiment, theprecipitate is a Cohn fraction IV-1 precipitate. In another embodiment,the precipitate is a Cohn fraction IV-4 precipitate. In anotherembodiment, the precipitate is a Cohn fraction V precipitate. In anotherembodiment, the precipitate is a Kistler/Nitschmann precipitate IV. Inyet another embodiment, the precipitate is a Kistler/Nitschmannprecipitate C.

2. Binding of Serine Proteases or Serine Protease Zymogens andExtraction of A1PI

In another aspect, the present invention provides a method for reducingthe amount of serine protease and/or serine protease zymogen in are-suspended plasma precipitate comprising A1PI. Generally, theprecipitate may be any precipitated during the fractionation of pooledplasma, preferably human plasma In one embodiment, the method comprisescontacting a re-suspended plasma precipitate comprising A1PI in aninsoluble state with finely divided silicon dioxide (SiO₂) under a firstsolution condition comprising low pH to bind the serine protease and/orserine protease zymogen and to maintain the A1PI in an insoluble state,separating the soluble and insoluble portions of the suspension,extracting the A1PI from the insoluble portion under a second solutioncondition comprising high pH, and separating the soluble portion fromthe insoluble portion, wherein a substantial fraction of the serineprotease and/or serine protease zymogen remains bound to the SiO₂ duringthe extraction of A1PI from the insoluble portion. In one embodiment,the SiO₂ is admixed prior to or during the precipitation reaction andrecovered along with the precipitate. In a specific embodiment, theprecipitate is a Cohn fraction IV-1 precipitate. In another embodiment,the precipitate is a Cohn fraction IV-4 precipitate. In anotherembodiment, the precipitate is a Cohn fraction V precipitate. In anotherembodiment, the precipitate is a Kistler/Nitschmann precipitate IV. Inyet another embodiment, the precipitate is a Kistler/Nitschmannprecipitate C.

In one embodiment of the methods provided above, the first solutioncondition comprises a pH of between 4.0 and 7.0 and an ionic strength ofless than about 5.0 mS/cm. In another embodiment, the solution conditioncomprises a pH of between 5.0 and 7.0 and an ionic strength of less thanabout 5.0 mS/cm. In another embodiment, the solution condition comprisesa pH of between 5.0 and 6.5 and an ionic strength of less than about 5.0mS/cm. In another embodiment, the solution condition comprises a pH ofbetween 5.0 and 6.0 and an ionic strength of less than about 5.0 mS/cm.In another embodiment, the solution condition comprises a pH of between5.5 and 6.0 and an ionic strength of less than about 5.0 mS/cm. In aspecific embodiment, the solution condition comprises a pH of 5.5±0.2and an ionic strength of less than about 5.0 mS/cm. In another specificembodiment, the solution condition comprises a pH of 6.0±0.2 and anionic strength of less than about 5.0 mS/cm.

In another embodiment of the methods provided above, the first solutioncondition comprises a pH of between 5.0 and 6.5 and an ionic strength ofless than about 4.0 mS/cm. In another embodiment, the solution conditioncomprises a pH of between 5.0 and 6.5 and an ionic strength of less thanabout 3.0 mS/cm. In another embodiment, the solution condition comprisesa pH of between 5.0 and 6.5 and an ionic strength of less than about 2.0mS/cm. In another embodiment, the solution condition comprises a pH ofbetween 5.0 and 6.5 and an ionic strength of less than about 1.0 mS/cm.In another embodiment, the solution condition comprises a pH of between5.0 and 6.5 and an ionic strength of less than about 0.5 mS/cm. Inanother embodiment, the solution condition comprises a pH of between 5.5and 6.0 and an ionic strength of less than about 4.0 mS/cm. In anotherembodiment, the solution condition comprises a pH of between 5.5 and 6.0and an ionic strength of less than about 3.0 mS/cm. In anotherembodiment, the solution condition comprises a pH of between 5.5 and 6.0and an ionic strength of less than about 2.0 mS/cm. In anotherembodiment, the solution condition comprises a pH of between 5.5 and 6.0and an ionic strength of less than about 1.0 mS/cm. In anotherembodiment, the solution condition comprises a pH of between 5.5 and 6.0and an ionic strength of less than about 0.5 mS/cm. In a specificembodiment, the solution conditions comprises a pH of 5.5±0.2 and anionic strength of less than about 3.0 mS/cm. In another specificembodiment, the solution condition comprises a pH of 6.0±0.2 and anionic strength of less than about 3.0 mS/cm. In yet other embodiments,the first low pH solution condition comprises a pH and ionic strengthaccording to any one of variations Var. 1222 to 3041, as presented inTable 12, Table 13, Table 14, and Table 15.

In one embodiment of the methods provided above, the second solutioncondition comprises a pH of between 7.0 and 10.0 and an ionic strengthof less than about 10.0 mS/cm. In another embodiment, the solutioncondition comprises a pH of between 7.0 and 9.0 and an ionic strength ofless than about 10.0 mS/cm. In another embodiment, the solutioncondition comprises a pH of between 7.0 and 8.5 and an ionic strength ofless than about 10.0 mS/cm. In another embodiment, the solutioncondition comprises a pH of between 7.5 and 8.5 and an ionic strength ofless than about 10.0 mS/cm. In another embodiment, the solutioncondition comprises a pH of between 7.5 and 8.0 and an ionic strength ofless than about 10.0 mS/cm. In a specific embodiment, the solutioncondition comprises a pH of 7.5±0.2 and an ionic strength of less thanabout 10.0 mS/cm. In another specific embodiment, the solution conditioncomprises a pH of 8.0±0.2 and an ionic strength of less than about 10.0mS/cm.

In another embodiment of the methods provided above, the second solutioncondition comprises a pH of between 7.0 and 8.5 and an ionic strength ofless than about 9.0 mS/cm. In another embodiment, the solution conditioncomprises a pH of between 7.0 and 8.5 and an ionic strength of less thanabout 8.0 mS/cm. In another embodiment, the solution condition comprisesa pH of between 7.0 and 8.5 and an ionic strength of less than about 7.0mS/cm. In another embodiment, the solution condition comprises a pH ofbetween 7.0 and 8.5 and an ionic strength of less than about 6.0 mS/cm.In another embodiment, the solution condition comprises a pH of between7.0 and 8.5 and an ionic strength of less than about 5 mS/cm. In anotherembodiment, the solution condition comprises a pH of between 7.0 and 8.5and an ionic strength of less than about 4.0 mS/cm. In anotherembodiment, the solution condition comprises a pH of between 7.0 and 8.5and an ionic strength of less than about 3.0 mS/cm. In anotherembodiment, the solution condition comprises a pH of between 7.0 and 8.5and an ionic strength of less than about 2 mS/cm. In another embodiment,the solution condition comprises a pH of between 7.0 and 8.5 and anionic strength of between 2 mS/cm and 10 mS/cm.

In another embodiment of the methods provided above, the second solutioncondition comprises a pH of between 7.5 and 8.0 and an ionic strength ofless than about 9.0 mS/cm. In another embodiment, the solution conditioncomprises a pH of between 7.5 and 8.0 and an ionic strength of less thanabout 8.0 mS/cm. In another embodiment, the solution condition comprisesa pH of between 7.5 and 8.0 and an ionic strength of less than about 7.0mS/cm. In another embodiment, the solution condition comprises a pH ofbetween 7.5 and 8.0 and an ionic strength of less than about 6.0 mS/cm.In another embodiment, the solution condition comprises a pH of between7.5 and 8.0 and an ionic strength of less than about 5 mS/cm. In anotherembodiment, the solution condition comprises a pH of between 7.5 and 8.0and an ionic strength of less than about 4.0 mS/cm. In anotherembodiment, the solution condition comprises a pH of between 7.5 and 8.0and an ionic strength of less than about 3.0 mS/cm. In anotherembodiment, the solution condition comprises a pH of between 7.5 and 8.0and an ionic strength of less than about 2 mS/cm. In another embodiment,the solution condition comprises a pH of between 7.5 and 8.5 and anionic strength of between 2 mS/cm and 10 mS/cm. In a specificembodiment, the solution condition comprises a pH of 7.5±0.2 and anionic strength of between 2 mS/cm and 10 mS/cm. In another specificembodiment, the solution condition comprises a pH of 8.0±0.2 and anionic strength of between 2 mS/cm and 10 mS/cm.

IV. Pharmaceutical Compositions

In one aspect, the present invention provides compositions ofplasma-derived proteins having reduced levels of serine protease and/orserine protease zymogen, which are prepared according to any of themethods described herein. In certain embodiments, these compositionswill be formulated for pharmaceutical administration (i.e.,pharmaceutical compositions). Generally, the plasma-derived bloodprotein compositions prepared according to the methods provided hereinwill have reduced amidolytic activity and will provide better safetyprofiles than existing plasma-derived biologics currently available. Ina preferred embodiment, the compositions provided herein will havereduced Factor XI, Factor XIa, Factor XII, and/or Factor XIIa content.

In one embodiment, the present invention provides a plasma-derivedprotein composition prepared by a method comprising the steps of: (a)contacting the composition with finely divided silicon dioxide (SiO₂)under conditions suitable to bind at least one serine protease or serineprotease zymogen; and (b) separating the SiO₂ from the composition toremove the bound serine protease or serine protease zymogen. In oneembodiment, the composition is formulated for pharmaceuticaladministration. In a specific embodiment, the composition is formulatedfor intravenous administration. In another specific embodiment, thecomposition is formulated for intramuscular administration. In anotherembodiment, the composition is formulated for subcutaneousadministration. In yet another embodiment, the composition is formulatedfor intraocular administration. In certain embodiments, the compositioncomprises a plasma-derived protein is selected from an immunoglobulin(Ig), albumin, alpha-1-antitrypsin (A1PI), butyrylcholinesterase, FactorH, a protein of the complement system, and an inter-alpha-trypsininhibitor (IαI) protein.

In certain embodiments, the compositions described above are prepared bya method further comprising the step of performing a first targetprotein enrichment step to form a first enriched composition, prior tocontacting the composition with finely divided silicon dioxide (SiO₂).In certain embodiments, the first target protein enrichment step isselected from a protein precipitation step (e.g., an alcoholfractionation step), an ultrafiltration/diafiltration step, and achromatographic step.

In one embodiment, the present invention provides a plasma-derivedprotein composition prepared by a method comprising the steps of: (a)forming a first enriched plasma-derived target protein composition bypartially precipitating protein in a starting material derived frompooled plasma; (b) contacting the first enriched composition with finelydivided silicon dioxide (SiO₂) under conditions suitable to bind atleast one serine protease or serine protease zymogen; and (c) separatingthe SiO₂ from the composition to remove the bound serine protease orserine protease zymogen. In one embodiment, the partial precipitation isachieved using alcohol. In one embodiment, the composition is formulatedfor pharmaceutical administration. In a specific embodiment, thecomposition is formulated for intravenous administration. In anotherspecific embodiment, the composition is formulated for intramuscularadministration. In another embodiment, the composition is formulated forsubcutaneous administration. In yet another embodiment, the compositionis formulated for intraocular administration. In certain embodiments,the composition comprises a plasma-derived protein is selected from animmunoglobulin (Ig), albumin, alpha-1-antitrypsin (A1PI),butyrylcholinesterase, Factor H, a protein of the complement system, andan inter-alpha-trypsin inhibitor (IαI) protein.

In one embodiment, the present invention provides a plasma-derivedprotein composition prepared by a method comprising the steps of: (a)forming a first enriched plasma-derived target protein composition byultrafiltering and/or diafiltering a starting material derived frompooled plasma; (b) contacting the first enriched composition with finelydivided silicon dioxide (SiO₂) under conditions suitable to bind atleast one serine protease or serine protease zymogen; and (c) separatingthe SiO₂ from the composition to remove the bound serine protease orserine protease zymogen. In one embodiment, the partial precipitation isachieved using alcohol. In one embodiment, the composition is formulatedfor pharmaceutical administration. In a specific embodiment, thecomposition is formulated for intravenous administration. In anotherspecific embodiment, the composition is formulated for intramuscularadministration. In another embodiment, the composition is formulated forsubcutaneous administration. In yet another embodiment, the compositionis formulated for intraocular administration. In certain embodiments,the composition comprises a plasma-derived protein is selected from animmunoglobulin (Ig), albumin, alpha-1-antitrypsin (A1PI),butyrylcholinesterase, Factor H, a protein of the complement system, andan inter-alpha-trypsin inhibitor (IαI) protein.

In one embodiment, the present invention provides a plasma-derivedprotein composition prepared by a method comprising the steps of: (a)forming a first enriched plasma-derived target protein composition bycontacting a starting material derived from pooled plasma with achromatographic resin; (b) contacting the first enriched compositionwith finely divided silicon dioxide (SiO₂) under conditions suitable tobind at least one serine protease or serine protease zymogen; and (c)separating the SiO₂ from the composition to remove the bound serineprotease or serine protease zymogen. In one embodiment, the partialprecipitation is achieved using alcohol. In one embodiment, thecomposition is formulated for pharmaceutical administration. In aspecific embodiment, the composition is formulated for intravenousadministration. In another specific embodiment, the composition isformulated for intramuscular administration. In another embodiment, thecomposition is formulated for subcutaneous administration. In yetanother embodiment, the composition is formulated for intraocularadministration. In certain embodiments, the composition comprises aplasma-derived protein is selected from an immunoglobulin (Ig), albumin,alpha-1-antitrypsin (A1PI), butyrylcholinesterase, Factor H, a proteinof the complement system, and an inter-alpha-trypsin inhibitor (IαI)protein.

In certain embodiments, the compositions described above are prepared bya method further comprising the step of performing a second targetprotein enrichment step to form a second enriched composition, prior tocontacting the composition with finely divided silicon dioxide (SiO₂).In certain embodiments, the first target protein enrichment step isselected from a protein precipitation step (e.g., an alcoholfractionation step), an ultrafiltration/diafiltration step, and achromatographic step.

In one embodiment, the present invention provides a plasma-derivedprotein composition prepared by a method comprising the steps of: (a)performing a first target protein enrichment step to form a firstenriched plasma-derived target protein composition; (b) performing asecond target protein enrichment step to form a second enrichedplasma-derived target protein composition; (c) contacting the secondenriched composition with finely divided silicon dioxide (SiO₂) underconditions suitable to bind at least one serine protease or serineprotease zymogen; and (d) separating the SiO₂ from the composition toremove the bound serine protease or serine protease zymogen. In certainembodiments, the combination of first and second enrichment steps isselected from any one of variations Var. 1 to Var. 100, found inTable 1. In one embodiment, the composition is formulated forpharmaceutical administration. In a specific embodiment, the compositionis formulated for intravenous administration. In another specificembodiment, the composition is formulated for intramuscularadministration. In another embodiment, the composition is formulated forsubcutaneous administration. In yet another embodiment, the compositionis formulated for intraocular administration. In certain embodiments,the composition comprises a plasma-derived protein is selected from animmunoglobulin (Ig), albumin, alpha-1-antitrypsin (A1PI),butyrylcholinesterase, Factor H, a protein of the complement system, andan inter-alpha-trypsin inhibitor (IαI) protein.

In certain embodiments, the compositions described above are prepared bya method further comprising the step of performing a target proteinenrichment step after contacting the composition with finely dividedsilicon dioxide (SiO₂). In certain embodiments, the target proteinenrichment step is selected from a protein precipitation step (e.g., analcohol fractionation step), an ultrafiltration/diafiltration step, anda chromatographic step.

In one embodiment, the present invention provides a plasma-derivedprotein composition prepared by a method comprising the steps of: (a)performing a first target protein enrichment step to form a firstenriched plasma-derived target protein composition; (b) contacting thefirst enriched composition with finely divided silicon dioxide (SiO₂)under conditions suitable to bind at least one serine protease or serineprotease zymogen; (c) separating the SiO₂ from the composition to removethe bound serine protease or serine protease zymogen; and (d) performinga second target protein enrichment step to form a second enrichedplasma-derived target protein composition. In certain embodiments, thecombination of first and second enrichment steps is selected from anyone of variations Var. 1 to Var. 100, found in Table 1. In oneembodiment, the composition is formulated for pharmaceuticaladministration. In a specific embodiment, the composition is formulatedfor intravenous administration. In another specific embodiment, thecomposition is formulated for intramuscular administration. In anotherembodiment, the composition is formulated for subcutaneousadministration. In yet another embodiment, the composition is formulatedfor intraocular administration. In certain embodiments, the compositioncomprises a plasma-derived protein is selected from an immunoglobulin(Ig), albumin, alpha-1-antitrypsin (A1PI), butyrylcholinesterase, FactorH, a protein of the complement system, and an inter-alpha-trypsininhibitor (IαI) protein.

In one aspect, the present invention provides a plasma-derived proteincomposition having reduced levels of serine protease and/or serineprotease zymogen for use in the treatment of a condition associated witha blood protein deficiency or dysfunction. In certain embodiments, theplasma-derived protein is selected from an immunoglobulin (Ig), albumin,alpha-1-antitrypsin (A1PI), butyrylcholinesterase, Factor H, a proteinof the complement system, and an inter-alpha-trypsin inhibitor (IαI)protein.

In one embodiment, the present invention provides a plasma-derivedprotein composition prepared by a method comprising the steps of: (a)performing a first target protein enrichment step to form a firstenriched plasma-derived target protein composition; (b) performing asecond target protein enrichment step to form a second enrichedplasma-derived target protein composition; (c) contacting the secondenriched composition with finely divided silicon dioxide (SiO₂) underconditions suitable to bind at least one serine protease or serineprotease zymogen; (d) separating the SiO₂ from the composition to removethe bound serine protease or serine protease zymogen; and (e) performinga third target protein enrichment step to form a third enrichedplasma-derived target protein composition. In certain embodiments, thecombination of first and second enrichment steps is selected from anyone of variations Var. 101 to Var. 1100, found in Table 2, Table 3,Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, or Table11. In one embodiment, the composition is formulated for pharmaceuticaladministration. In a specific embodiment, the composition is formulatedfor intravenous administration. In another specific embodiment, thecomposition is formulated for intramuscular administration. In anotherembodiment, the composition is formulated for subcutaneousadministration. In yet another embodiment, the composition is formulatedfor intraocular administration. In certain embodiments, the compositioncomprises a plasma-derived protein is selected from an immunoglobulin(Ig), albumin, alpha-1-antitrypsin (A1PI), butyrylcholinesterase, FactorH, a protein of the complement system, and an inter-alpha-trypsininhibitor (IαI) protein.

In certain embodiments of the compositions described above, achromatographic enrichment step comprises the sub-steps of: (i)contacting the plasma-derived target protein composition with achromatographic resin under conditions suitable to bind theplasma-derived target protein; and (ii) eluting the plasma-derivedtarget protein from the chromatographic resin. In one specificembodiment, the impurity does not bind to the chromatographic resin insub-step (i). In another specific embodiment, the impurity binds to thechromatographic resin in sub-step (i), but is not eluted from thechromatographic resin in sub-step (ii).

In other certain embodiments of the compositions described above, achromatographic enrichment step comprises the sub-steps of: (i)contacting the first enriched plasma-derived target protein compositionwith a chromatographic resin under conditions suitable to bind at leastone impurity; and (ii) separating the resin from the plasma-derivedprotein composition, wherein the plasma-derived target protein does notbind to the chromatographic resin in sub-step (i).

In certain embodiments of the compositions provided herein, the amountof a particular serine protease or serine protease zymogen is reduced byat least 10%. In another embodiment, the amount of a particular serineprotease or serine protease zymogen is reduced by at least 25%. Inanother embodiment, the amount of a particular serine protease or serineprotease zymogen is reduced by at least 50%. In another embodiment, theamount of a particular serine protease or serine protease zymogen isreduced by at least 75%. In another embodiment, the amount of aparticular serine protease or serine protease zymogen is reduced by atleast 90%. In yet other embodiments, the amount of a particular serineprotease or serine protease zymogen is reduced by at least 5%, or by atleast 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Inone embodiment, the reduction of serine protease or serine proteasezymogen refers to the reduction achieved within the individual SiO₂treatment step. In another embodiment, the reduction of serine proteaseor serine protease zymogen refers to the level of the contaminant in thefinal composition, as compared to a composition prepared in a similarfashion excluding a SiO₂ treatment step.

In one embodiment, the pharmaceutical compositions provided herein areprepared by formulating a plasma-derived protein composition isolatedusing a method provided herein. Generally, the formulated compositionwill have been subjected to at least one, preferably at least two, mostpreferably at least three, viral inactivation or removal steps.Non-limiting examples of viral inactivation or removal steps that may beemployed with the methods provided herein include, solvent detergenttreatment (Horowitz et al., Blood Coagul Fibrinolysis 1994 (5 Suppl3):S21-S28 and Kreil et al., Transfusion 2003 (43):1023-1028, both ofwhich are herein expressly incorporated by reference in their entiretyfor all purposes), nanofiltration (Hamamoto et al., Vox Sang 1989(56)230-236 and Yuasa et al., J Gen Virol. 1991 (72 (pt 8)):2021-2024,both of which are herein expressly incorporated by reference in theirentirety for all purposes), and low pH incubation at high temperatures(Kempf et al., Transfusion 1991 (31)423-427 and Louie et al.,Biologicals 1994 (22):13-19). In certain embodiments, the compositioncomprises a plasma-derived protein is selected from an immunoglobulin(Ig), albumin, alpha-1-antitrypsin (A1PI), butyrylcholinesterase, FactorH, a protein of the complement system, and an inter-alpha-trypsininhibitor (IαI) protein.

In one embodiment, the pharmaceutical compositions provided herein willcomprise one or more buffering agents or pH stabilizing agents suitablefor intravenous, subcutaneous, intramuscular, and/or intraocularadministration. Non-limiting examples of buffering agents suitable forformulating a plasma-derived protein composition provided herein includeglycine, citrate, phosphate, acetate, glutamate, tartrate, benzoate,lactate, histidine or other amino acids, gluconate, malate, succinate,formate, propionate, carbonate, or any combination thereof adjusted toan appropriate pH. Generally, the buffering agent will be sufficient tomaintain a suitable pH in the formulation for an extended period oftime. In a preferred embodiment, the buffering agent is glycine. Incertain embodiments, the composition comprises a plasma-derived proteinis selected from an immunoglobulin (Ig), albumin, alpha-1-antitrypsin(A1PI), butyrylcholinesterase, Factor H, a protein of the complementsystem, and an inter-alpha-trypsin inhibitor (IαI) protein.

In some embodiments, the pharmaceutical compositions provided herein mayoptionally further comprise an agent for adjusting the osmolarity of thecomposition. Non-limiting examples of osmolarity agents includemannitol, sorbitol, glycerol, sucrose, glucose, dextrose, levulose,fructose, lactose, polyethylene glycols, phosphates, sodium chloride,potassium chloride, calcium chloride, calcium gluconoglucoheptonate,dimethyl sulfone, and the like.

Typically, the formulations provided herein will have osmolarities thatare comparable to physiologic osmolarity, about 285 to 295 mOsmol/kg(Lacy et al., Drug Information Handbook—Lexi-Comp 1999:1254. In certainembodiments, the osmolarity of the formulation will be between about 200mOsmol/kg and about 350 mOsmol/kg, preferably between about 240 andabout 300 mOsmol/kg. In particular embodiments, the osmolarity of theformulation will be about 200 mOsmol/kg, or 210 mOsmol/kg, 220mOsmol/kg, 230 mOsmol/kg, 240 mOsmol/kg, 245 mOsmol/kg, 250 mOsmol/kg,255 mOsmol/kg, 260 mOsmol/kg, 265 mOsmol/kg, 270 mOsmol/kg, 275mOsmol/kg, 280 mOsmol/kg, 285 mOsmol/kg, 290 mOsmol/kg, 295 mOsmol/kg,300 mOsmol/kg, 310 mOsmol/kg, 320 mOsmol/kg, 330 mOsmol/kg, 340mOsmol/kg, 340 mOsmol/kg, or 350 mOsmol/kg.

The plasma-derived formulations provided herein are generally stable inliquid form for an extended period of time. In certain embodiments, theformulations are stable for at least about 3 months at room temperature,or at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, or 24 months at room temperature. The formulationwill also generally be stable 6 or at least about 18 months underrefrigerated conditions (typically between about 2° C. and about 8° C.),or for at least about 21, 24, 27, 30, 33, 36, 39, 42, or 45 months underrefrigerated conditions.

V. Methods of Treatment

In one aspect, the present invention provides methods for treating adisease or disorder associated with a blood protein deficiency ordysfunction in a subject in need thereof by administering atherapeutically effective dose of a plasma-derived protein compositionhaving reduced levels of serine protease and/or serine protease zymogenprepared according to a method provided herein. In certain embodiments,the composition comprises a plasma-derived protein is selected from animmunoglobulin (Ig), albumin, alpha-1-antitrypsin (A1PI),butyrylcholinesterase, Factor H, a protein of the complement system, andan inter-alpha-trypsin inhibitor (IαI) protein.

In one aspect, the present invention provides the use of aplasma-derived protein composition having reduced levels of serineprotease and/or serine protease zymogen for the manufacture of amedicament for use in the treatment of a condition associated with ablood protein deficiency or dysfunction. In certain embodiments, theplasma-derived protein is selected from an immunoglobulin (Ig), albumin,alpha-1-antitrypsin (A1PI), butyrylcholinesterase, Factor H, a proteinof the complement system, and an inter-alpha-trypsin inhibitor (IαI)protein.

A. Immunoglobulins

As routinely practiced in the modern medicine, sterilized preparationsof concentrated immunoglobulins (especially IgGs) are used for treatingmedical conditions that fall into these three main classes: immunedeficiencies, inflammatory and autoimmune diseases, and acuteinfections. These IgG preparations may also be useful for treatingmultiple sclerosis (especially relapsing-remitting multiple sclerosis orRRMS), Alzheimer's disease, and Parkinson's disease. The purified IgGpreparation of this invention is suitable for these purposes, as well asother clinically accepted uses of IgG preparations.

The FDA has approved the use of IVIG to treat various indications,including allogeneic bone marrow transplant, chronic lymphocyticleukemia, idiopathic thrombocytopenic purpura (ITP), pediatric HIV,primary immunodeficiencies, Kawasaki disease, chronic inflammatorydemyelinating polyneuropathy (CIDP), and kidney transplant with a highantibody recipient or with an ABO incompatible donor. In certainembodiments, the IVIG compositions provided herein are useful for thetreatment or management of these diseases and conditions.

Furthermore, off-label uses for IVIG are commonly provided to patientsfor the treatment or management of various indications, for example,chronic fatigue syndrome, clostridium difficile colitis, dermatomyositisand polymyositis, Graves' ophthalmopathy, Guillain-Barré syndrome,muscular dystrophy, inclusion body myositis, Lambert-Eaton syndrome,Lupus erythematosus, multifocal motor neuropathy, multiple sclerosis(MS), myasthenia gravis, neonatal alloimmune thrombocytopenia,Parvovirus B19 infection, pemphigus, post-transfusion purpura, renaltransplant rejection, spontaneous Abortion/Miscarriage, stiff personsyndrome, opsoclonus Myoclonus, severe sepsis and septic shock incritically ill adults, toxic epidermal necrolysis, chronic lymphocyticleukemia, multiple myeloma, X-linked agammaglobulinemia, andhypogammaglobulinemia. In certain embodiments, the IVIG compositionsprovided herein are useful for the treatment or management of thesediseases and conditions.

Finally, experimental use of IVIG for the treatment or management ofdiseases including primary immune deficiency, RRMS, Alzheimer's disease,and Parkinson's disease has been proposed (U.S. Patent ApplicationPublication No. U.S. 2009/0148463, which is herein incorporated byreference in its entirety for all purposes). In certain embodiments, theIVIG compositions provided herein are useful for the treatment ormanagement of primary immune deficiency, RRMS, Alzheimer's disease, orParkinson's disease. In certain embodiments comprising dailyadministration, an effective amount to be administered to the subjectcan be determined by a physician with consideration of individualdifferences in age, weight, disease severity, route of administration(e.g., intravenous v. subcutaneous) and response to the therapy. Incertain embodiments, an immunoglobulin preparation of this invention canbe administered to a subject at about 5 mg/kilogram to about 2000mg/kilogram each day. In additional embodiments, the immunoglobulinpreparation can be administered in amounts of at least about 10mg/kilogram, at last 15 mg/kilogram, at least 20 mg/kilogram, at least25 mg/kilogram, at least 30 mg/kilogram, or at least 50 mg/kilogram. Inadditional embodiments, the immunoglobulin preparation can beadministered to a subject at doses up to about 100 mg/kilogram, to about150 mg/kilogram, to about 200 mg/kilogram, to about 250 mg/kilogram, toabout 300 mg/kilogram, to about 400 mg/kilogram each day. In otherembodiments, the doses of the immunoglobulin preparation can be greateror less. Further, the immunoglobulin preparations can be administered inone or more doses per day. Clinicians familiar with the diseases treatedby IgG preparations can determine the appropriate dose for a patientaccording to criteria known in the art.

In accordance with the present invention, the time needed to complete acourse of the treatment can be determined by a physician and may rangefrom as short as one day to more than a month. In certain embodiments, acourse of treatment can be from 1 to 6 months.

An effective amount of an IVIG preparation is administered to thesubject by intravenous means. The term “effective amount” refers to anamount of an IVIG preparation that results in an improvement orremediation of disease or condition in the subject. An effective amountto be administered to the subject can be determined by a physician withconsideration of individual differences in age, weight, the disease orcondition being treated, disease severity and response to the therapy.In certain embodiments, an IVIG preparation can be administered to asubject at dose of about 5 mg/kilogram to about 2000 mg/kilogram peradministration. In certain embodiments, the dose may be at least about 5mg/kg, or at least about 10 mg/kg, or at least about 20 mg/kg, 30 mg/kg,40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg,125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg,700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, 1000mg/kg, 1100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, or at least about 2000 mg/kg.

The dosage and frequency of IVIG treatment will depend upon, among otherfactors. the disease or condition being treated and the severity of thedisease or condition in the patient. Generally, for primary immunedysfunction a dose of between about 100 mg/kg and about 400 mg/kg bodyweight will be administered about every 3 to 4 weeks. For neurologicaland autoimmune diseases, up to 2 g/kg body weight is implemented forthree to six months over a five day course once a month. This isgenerally supplemented with maintenance therapy comprising theadministration of between about 100 mg/kg and about 400 mg/kg bodyweight about once every 3 to 4 weeks. Generally, a patient will receivea dose or treatment about once every 14 to 35 days, or about every 21 to28 days. The frequency of treatment will depend upon, among otherfactors. the disease or condition being treated and the severity of thedisease or condition in the patient.

In a preferred embodiment, a method of treating an immunodeficiency,autoimmune disease, or acute infection in a human in need thereof isprovided, the method comprising administering a pharmaceutical IVIGcomposition of the present invention. In a related embodiment, thepresent invention provides IVIG compositions manufactured according to amethod provided herein for the treatment of an immunodeficiency,autoimmune disease, or acute infection in a human in need thereof.

In certain embodiments, the immunodeficiency, autoimmune disease, oracute infection is selected from allogeneic bone marrow transplant,chronic lymphocytic leukemia, idiopathic thrombocytopenic purpura (ITP),pediatric HIV, primary immunodeficiencies, Kawasaki disease, chronicinflammatory demyelinating polyneuropathy (CIDP), kidney transplant witha high antibody recipient or with an ABO incompatible donor, chronicfatigue syndrome, clostridium difficile colitis, dermatomyositis andpolymyositis, Graves' ophthalmopathy, Guillain-Barré syndrome, musculardystrophy, inclusion body myositis, Lambert-Eaton syndrome, Lupuserythematosus, multifocal motor neuropathy, multiple sclerosis (MS),myasthenia gravis, neonatal alloimmune thrombocytopenia, Parvovirus B19infection, pemphigus, post-transfusion purpura, renal transplantrejection, spontaneous Abortion/Miscarriage, stiff person syndrome,opsoclonus Myoclonus, severe sepsis and septic shock in critically illadults, toxic epidermal necrolysis, chronic lymphocytic leukemia,multiple myeloma, X-linked agammaglobulinemia, hypogammaglobulinemia,primary immune deficiency, RRMS, Alzheimer's disease, and Parkinson'sdisease.

B. Factor H

In one aspect, the present invention provides methods for treating adisease or disorder associated with a Factor H dysfunction or abnormalalternative pathway complement activity in a subject in need thereof byadministering a therapeutically effective dose of a Factor H compositionprepared according to a method provided herein. In one embodiment, theFactor H composition is prepared by extracting Factor H from a FractionI precipitate. In another embodiment, the Factor H composition isprepared by extracting Factor H from a Fraction II+III filter cake.

In certain embodiments, the disease or disorder associated with a FactorH dysfunction is selected from atypical haemolytic uremic syndrome(aHUS), age-related macular degeneration (AMD), membranoproliferativeglomulonephritis type II (MPGNII), myocardial infarction, coronary heartdisease/coronary artery disease (CAD/CHD), and Alzheimer's disease. Inone particular embodiment, the disease is atypical haemolytic uremicsyndrome (aHUS). In another particular embodiment, the disease isage-related macular degeneration (AMD). In yet another particularembodiment, the disease is membranoproliferative glomulonephritis typeII (MPGNII).

In certain embodiments, a method is provided for treating a disease ordisorder associated with a abnormal alternative pathway complementactivity in a subject in need thereof by administering to the subject atherapeutically effective dose of a Factor H composition providedherein. In one embodiment, the Factor H composition is prepared byextracting Factor H from a Fraction I precipitate. In anotherembodiment, the Factor H composition is prepared by extracting Factor Hfrom a Fraction II+III filter cake.

In certain embodiments, the disease or disorder associated with abnormalalternative pathway complement activity is selected from an autoimmunedisease (such as rheumatoid arthritis, IgA nephropathy, asthma, systemiclupus erythematosus, multiple sclerosis, Anti-Phospholipid syndrome,ANCA-associated vasculitis, pemphigus, uveitis, myathemia gravis,Hashimoto's thyroiditis), a renal disease (such as IgA nephropathy,hemolytic uremic syndrome, membranoproliferative glomerulonephritis)asthma, Alzheimer disease, adult macular degeneration, proximalnocturnal hemoglobinuria, abdominal aortic aneurism, ischemiareperfusion injury, and sepsis.

The pharmaceutical compositions provided by the invention may beadministered alone or in conjunction with other therapeutic agents.These agents may be incorporated as part of the same pharmaceutical.

1. Administration

In accordance with the present invention, the time needed to complete acourse of the treatment can be determined by a physician and may rangefrom as short as one day to more than a month. In certain embodiments, acourse of treatment can be from 1 to 6 months.

An effective amount of a Factor H preparation is administered to thesubject by any suitable means to treat the disease or disorder. Forexample, in certain embodiments, Factor H may be administered byintravenous, intraocular, subcutaneous, and/or intramuscular means. In apreferred embodiment, a method for treating age-related maculardegeneration in a subject in need thereof is provided comprising theintraocular administration of a Factor H composition to the patient.

In certain embodiments, the Factor H compositions provided herein can beadministered either systemically or locally. Systemic administrationincludes: oral, transdermal, subdermal, intraperitioneal, subcutaneous,transnasal, sublingual, or rectal. The most preferred systemic route ofadministration is oral. Local administration for ocular administrationincludes: topical, intravitreal, periocular, transcleral, retrobulbar,juxtascleral, sub-tenon, or via an intraocular device. Preferred methodsfor local delivery include transscleral delivery to the macula byposterior juxtascleral administration; via intravitreal injection; orvia cannula, such as that described in U.S. Pat. No. 6,413,245, thedisclosure of which is incorporated by reference herein in its entiretyfor all purposes. Alternatively, the inhibitors may be delivered via asustained delivery device implanted intravitreally or transsclerally, orby other known means of local ocular delivery.

In certain embodiments, the term “effective amount” refers to an amountof a Factor H preparation that results in an improvement or remediationof disease or condition in the subject. An effective amount to beadministered to the subject can be determined by a physician withconsideration of individual differences in age, weight, the disease orcondition being treated, disease severity and response to the therapy.In certain embodiments, an Factor H preparation can be administered to asubject at dose of at or about between 5 mg/kilogram and 2000mg/kilogram per administration. In certain embodiments, the dose may beat least at or about 5 mg/kg, or at least at or about 10 mg/kg, or atleast at or about 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg,200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg,850 mg/kg, 900 mg/kg, 950 mg/kg, 1000 mg/kg, 1100 mg/kg, 1200 mg/kg,1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg,1900 mg/kg, or 2000 mg/kg. The dosage and frequency of Factor Htreatment will depend upon, among other factors, the disease orcondition being treated and the severity of the disease or condition inthe patient.

2. Age-Related Macular Degeneration (AMD)

In a preferred embodiment, the present invention provides a method oftreating age-related macular degeneration in a subject in need thereofby administering to the subject a therapeutically effective dose of aFactor H composition provided herein.

Age-related macular degeneration (AMD) is the number one cause ofblindness for the elderly population over 60 years of age. Today, it isestimated that 35-40% of those over 75 years of age have some degree ofAMD. It has been estimated that approximately 50 million people areaffected world-wide, with 10 million in the US alone. Currently, about155,000 new diagnoses of AMD are made every year. As the worldwidepopulation continues to age, the number of annual diagnoses are expectedto triple by the year 2020. It is a devastating disease that destroyscentral vision in the affected individuals, robbing them of theirability to perform activities necessary for everyday life such asreading and driving.

AMD is a slow, progressive disease that involves cells of the outerretinal layers (including photoreceptors and the retinal pigmentepithelial (RPE) cells that support the photoreceptors), as well ascells in the adjacent vascular layer of the eye known as the choroid.Macular degeneration is characterized by the breakdown of the macula, asmall portion of the central retina (about 2 mm in diameter) responsiblefor high-acuity vision. Late-onset macular degeneration (i.e., AMD) isgenerally defined as either “dry” or “wet.” The wet (“exudative”)neovascular form of AMD affects approximately 10% of those with thedisease, and is characterized by abnormal blood vessels growing from thechoriocapillaris through the RPE, typically resulting in hemorrhage,exudation, scarring, and/or serous retinal detachment. Approximately 90%of patients with AMD have the non-neovascular, or dry form of thedisease, which is characterized by atrophy of the RPE and loss ofmacular photoreceptors.

AMD is characterized by the presence of deposits of debris-likematerial, termed “drusen,” that accumulate on Bruch's membrane, amultilayered composite of extracellular matrix components separating theRPE (the outermost layer of the retina) from the underlying choroid.Drusen can be observed by funduscopic eye examination. These depositshave been extensively characterized in microscopic studies of donor eyesfrom patients with AMD. The deposits observed in the living eye uponclinical examination are classified as either soft drusen or harddrusen, according to several criteria including relative size,abundance, and shape of the deposits. Histochemical andimmunocytochemical studies have shown that drusen contain a variety oflipids, polysaccharides, glycosaminoglycans and proteins.

Presently, there no known cure for AMD, although several types oftreatments has been shown to be effective at managing the disease. Laserphotocoagulation of abnormal vessels in the wet form of the disease isthe standard treatment. This treatment is limited by the fact that onlywell-delineated neovascular lesions can be treated in this way and that50% of patients will suffer recurrence of the leakage from the vessels(Fine et al., 2000). Because of the energy of the laser required forthis treatment, the photoreceptors in the treated area will also die,and the patient will also often suffer central blindness immediatelyafter the treatment. New neovascular lesions will eventually develop,requiring repeated treatments. Other interventions include changinglifestyles by cessation of smoking and beginning therapy withantioxidants. Antiangiogenic treatments using VEGF inhibitors e.g.,intravitrial injection of ranibizumab or bevacizumab also have beensuggested.

Recently it was discovered that about 35% of individuals carry at anat-risk single nucleotide polymorphism (SNP) in one or both copies oftheir Factor H gene. Homozygous individuals have an approximatelysevenfold increased chance of developing age-related maculardegeneration, while heterozygotes have a two-to-threefold increasedlikelihood of developing the disease. This SNP, located in CCP module 7of Factor H, has been shown to affect the interactions between Factor Hand both C-reactive protein and heparin indicating a causal relationshipbetween the SNP and disease. The polymorphism is a Y420H polymorphism.

In one embodiment of a method for limiting complement activationresulting in delayed progression or onset of the development of agerelated macular degeneration (AMD) in a subject, the subject does nothave any symptoms of AMD.

In another embodiment of a method for limiting complement activationresulting in delayed progression or onset of the development of agerelated macular degeneration (AMD) in a subject, the subject has drusen.

In another embodiment of a method for limiting complement activationresulting in delayed progression or onset of the development of agerelated macular degeneration (AMD) in a subject, the subject is atincreased risk of developing AMD.

In another embodiment of a method for limiting complement activationresulting in delayed progression or onset of the development of agerelated macular degeneration (AMD) in a subject, the administration isintravenous.

In another embodiment of a method for limiting complement activationresulting in delayed progression or onset of the development of agerelated macular degeneration (AMD) in a subject, the method furthercomprises treating a subject having signs and/or symptoms of AMD.

In another embodiment of a method for limiting complement activationresulting in delayed progression or onset of the development of agerelated macular degeneration (AMD) in a subject, the subject has beendiagnosed with AMD.

In another aspect, the present invention provides a method of treating ahuman subject judged to be at risk for the development of age relatedmacular degeneration, comprising the step of administering to thesubject a prophylactically or therapeutically effective amount of aFactor H preparation provided herein, and periodically repeating saidadministration.

In one embodiment of a method of treating a human subject judged to beat risk for the development of age related macular degeneration, theadministration is repeated for a time effective to delay the progressionor onset of the development of macular degeneration in said subject.

In another embodiment of a method of treating a human subject judged tobe at risk for the development of age related macular degeneration, thehuman subject is judged to be at risk for the development of age-relatedmacular degeneration as identified based on the presence of one or moregenetic markers associated with development of age-related maculardegeneration.

In another embodiment of a method of treating a human subject judged tobe at risk for the development of age related macular degeneration, thegenetic marker is a polymorphism.

In another embodiment of a method for limiting complement activationresulting in delayed progression or onset of the development of agerelated macular degeneration (AMD) in a subject, the subject is notdiagnosed with AMD.

C. Inter-alpha-Trypsin Inhibitor (IαI)

In yet other aspects, it is an object of the invention to providemethods for treating disorders and diseases associated with reduced IaIpfunction or IaIp dysfunction by administering a therapeuticallyeffective amount of an IaIp composition provided herein. In oneembodiment, the disease or disorder associated with reduced IaIpfunction or IaIp dysfunction is sepsis.

In one embodiment, the present invention provides a therapeuticallyeffective dose of an IaIp composition prepared by a method disclosedherein for use in a method for treating a disease or disorder associatedwith reduced IaIp function or IaIp dysfunction in a subject in needthereof. In one embodiment, the disease or disorder associated withreduced IaIp function or IaIp dysfunction is sepsis.

In another aspect, it is an object of the invention to provide methodsfor treating diseases and disorders associated with increased plasmaserine protease activity by administering a therapeutically effectiveamount of an IaIp composition provided herein. In one embodiment, thedisease or disorder associated increased plasma serine protease activityis selected from sepsis, septic shock, endotoxic shock, disseminatedintravascular coagulation, fibroproliferation, anthrax intoxication,cancer metastasis, tissue injury during surgery, kidney disease,vascular disease, coagulation, diabetes, and systemic inflammation.

In one embodiment, the present invention provides a therapeuticallyeffective dose of an IaIp composition prepared by a method disclosedherein for use in a method for treating a disease or disorder associatedwith increased plasma serine protease activity in a subject in needthereof. In one embodiment, the disease or disorder associated increasedplasma serine protease activity is selected from sepsis, septic shock,endotoxic shock, disseminated intravascular coagulation,fibroproliferation, anthrax intoxication, cancer metastasis, tissueinjury during surgery, kidney disease, vascular disease, coagulation,diabetes, and systemic inflammation.

A. Administration

In accordance with the present invention, the time needed to complete acourse of the treatment can be determined by a physician and may rangefrom as short as one day to more than a month. In certain embodiments, acourse of treatment can be from 1 to 6 months.

An effective amount of an IaIp preparation is administered to thesubject by any suitable means to treat the disease or disorder. Forexample, in certain embodiments, IaIp may be administered byintravenous, subcutaneous, and/or intramuscular means. In a preferredembodiment, a method for treating sepsis in a subject in need thereof isprovided comprising the intravenous (IV) administration of an IaIpcomposition to the patient.

In certain embodiments, the IaIp compositions provided herein can beadministered either systemically or locally. Systemic administrationincludes: oral, subdermal, intraperitioneal, subcutaneous, transnasal,sublingual, or rectal routes of administration. Local administrationincludes: topical, subcutaneous, intramuscular, and intraperitonealroutes of administration.

In certain embodiments, the term “effective amount” refers to an amountof a IaIp preparation that results in an improvement or remediation ofdisease or condition in the subject. An effective amount to beadministered to the subject can be determined by a physician withconsideration of individual differences in age, weight, the disease orcondition being treated, disease severity and response to the therapy.In certain embodiments, an IaIp preparation can be administered to asubject at dose of about 5 mg/kilogram to about 2000 mg/kilogram peradministration. In certain embodiments, the dose may be at least about 5mg/kg, or at least about 10 mg/kg, or at least about 20 mg/kg, 30 mg/kg,40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg,125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg,700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, 1000mg/kg, 1100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, or at least about 2000 mg/kg.The dosage and frequency of IaIp treatment will depend upon, among otherfactors, the disease or condition being treated and the severity of thedisease or condition in the patient.

VI. EXAMPLES Example 1

In order to determine the residual serine protease content and activitypresent in plasma-derived protein compositions, the amidolytic activityprofile was determined for two commercially available IgG preparationsthat were manufactured without the use of SiO₂ treatment: OCTAGAM® (5%Intravenous Immune Globulin; Octapharma) and Subcuvia (16% SubcutaneousImmune Globulin; Baxter); two lots of a commercially available IgGpreparation manufactured using SiO₂ treatment: Gammagard Liquid (10%Intravenous Immune Globulin; Baxter), and a Factor H purification methodcurrently in development. Notably, the Factor H composition was purifiedas described above, by binding and subsequently eluting Factor H fromfinely divided SiO₂.

Briefly, the amidolytic activity profile for each of the plasma-derivedprotein compositions was determined by assaying with the followingchromogenic substrates with different enzyme specificities: PL-1 (broadspectrum), S-2288 (broad spectrum), S-2266 (FXIa, glandularkallikreins), S-2222 (FXa, trypsin), S-2251 (Plasmin), and S-2302(Kallikrein, FXIa, and FXIIa). Pre-kallikrein activator activity (PKKA)and amount of Factor XIa Units was also determined. As shown in Table17, the plasma-derived IgG compositions manufactured without the use ofan SiO₂ adsorption step contained significant levels of amidolyticactivity and FXIa content. In contrast, both tested lots of theGammagard Liquid contained minimal amidolytic activity and FXIa content.Consistent with these results, the Factor H composition prepared bybinding and eluting from finely divided SiO₂, contains extremely highlevels of amidolytic activity and FXIa content.

TABLE 17 Amidolytic activity of various plasma-derived proteincompositions. Commercially available IGIV preparations Octagam 5%Gammagard Gammagard IGSC Factor H (Octa- Liquid 10% lot 1 Liquid 10% lot2 Subcuvia Sample FH012 pharma) (Baxter) (Baxter) 16% (Baxter)Chromogenic FC steril #B82A8432 #LE12G142AD #LE12HE76 #VNG1H020Specificity substrate Hydrolysis rate [nmol/ml × min] Broad PL-1 73.718.3 <10 <10 22.1 spectrum Broad S-2288 241 29 <5 <5 46 spectrum FXIa,glandular S-2266 171 27.1 <5 <5 34.2 kallikreins FXa, Trypsin S-2222 8.3<5 <5 <5 <5 Plasmin S-2251 7.3 <5 <5 <5 <5 Kallikrein, FXIa, S-2302 56370.1 <5 7.6 99.6 FXIIa PKKA IE/mL 9.5 <4 <4 <4 <4 F-XIa mU/mL 510.8 1.37<0.04 <0.04 0.79

Example 2

To determine an economically beneficial scheme for the manufacture ofFactor H from a plasma sample, which allows for the recovery ofadditional blood factors from the same plasma sample, a lot of pooledhuman plasma was subjected to fractionation according to the schemeoutlined in the flow-diagram shown in FIG. 1. As shown in FIG. 2, themajority of Factor H (about 90%) present in a human plasma cryo-poorCohn pool can be found in the fraction II+III precipitate. A smaller,yet significant, amount of Factor H (about 10%) can also be found in thefraction I precipitate. This is consistent with the results shown in PCTPublication No. WO 2011/011753, the contents of which are herebyincorporated by reference in their entirety for all purposes.

Factor H was extracted from the finely divided SiO₂ filter cakebyproduct formed as a result of filtering the “Aerosil Treatment”composition directly upstream of composition 6, the “Fraction II+IIIFiltrate,” by re-circulating a Factor H extraction buffer through thefilter press. Salts and various impurities were then removed from thefilter cake extract by a first precipitation step performed at pH 8.0via addition of ethanol to a final concentration of 15% and incubationat −6° C. for a minimum of four hours. The pH of the precipitationreaction was readjusted to 8.0 after 1 hour of incubation time. Theprecipitate was then removed from the supernatant by centrifugation.Factor H was further enriched by a second precipitation step performedat pH 6.0 via addition of ethanol to a final concentration of 25% andincubation at −10° C. for a minimum of 8 hours. The precipitatecontaining Factor H was then recovered by centrifugation.

The precipitate formed by the second precipitation step was dissolved ata ratio of 1:9 in a low ionic strength dissolution buffer and S/Dtreated to inactivate lipid enveloped viruses. Factor H was subsequentlyenriched by anion exchange chromatography using a DEAE-Sepharose FFresin. Briefly, Factor H was bound to DEAE-Sepharose resin under lowionic strength conditions and eluted by increasing the ionic strength ofthe solution. The conductivity of the DEAE-Sepharose eluate was thenreduced and Factor H was further enriched by Heparin-affinitychromatography. Briefly, Factor H was bound to Heparin-Sepharose FFresin under low ionic strength conditions and eluted by increasing theionic strength of the solution. As shown in Table 18, the majority ofFactor H bound to the DEAE and Heparin resins.

TABLE 18 Binding of Factor H to chromatographic resins. 1. DEAE- 2.Heparin- Sepharose FF Sepharose FF LOT FH006 FH012 FH006 FH012 Loading30.6 28.0 3.3 2.1 (Protein) mg/ml mg/ml mg/ml mg/ml FH binding 87.4%96.3% 100% 99.4% to resine

Factor H eluted from the Heparin resin was then subjected toultrafiltration/diafiltration according to standard procedures, followedby size exclusion chromatography on a Superdex 200 column. Factor Hrecovered from the size exclusion chromatography was then concentratedby ultrafiltration, sterile filtered, and formulated at a final proteinconcentration of 50 mg/mL in PBS-buffer.

The final Factor H composition (FH012) was then characterized forhomogeneity, impurities, and amidolytic activity. The monodispersity ofthe Factor H composition was characterized by size exclusionchromatography. As shown in Table 19, the majority of the proteinpresent in the Factor H final composition migrated with an estimatedsize of 400 kDa when loaded onto an HP-SEC column.

TABLE 19 Molecular size distribution of final FH012 composition asdetermined by HP-SEC. Peak 1 Peak 2 Peak 3 sample >450 kDa 400 kDa 160kDa % area FC FH012 0.3 97.6 2.1

The level of endotoxins, pH, visual appearance, and final proteinconcentration was then determined for the final Factor H composition. Asshown in Table 20, the composition had low endotoxin levels (<0.5 EU/mL)as determined by limulus amebocyte lysate (LAL) assay.

TABLE 20 LAL, pH, visual appearance, and protein content of the finalFH012 composition. LAL <0.5 EU/mL (pyrogen free) pH 7.1 VisualAppearance Colorless and free of visual particles Protein Concentration4.54%

The level of various protein impurities in the final Factor Hcomposition was then determined. As shown in Table 21, complementproteins and IgG immunoglobulins accounted for less than 1% of the finalprotein concentration in the Factor H composition.

TABLE 21 Impurities in the final FH012 composition. Percentage ofImpurity Concentration Total Protein IgG 51 μg/mL 0.11% C3 321.5 μg/mL0.71% C3a 17.5 μg/mL 0.04% C5a 3.7 ng/mL <0.01% C4 1.94 μg/mL <0.01%EDTA 72 μg/mL

Finally, the level of amidolytic activity and protease content wasdetermined as reported in Example 1. As shown in Table 17,plasma-derived Factor H purified according to the scheme outlined inthis example contained high levels of amidolytic activity and FXIacontent.

Example 3

In order to show the capability of removing amidolytic activity from aplasma-derived protein composition, re-suspended Cohn Fraction II+IIIprecipitates were treated with finely divided silicon dioxide (SiO₂).Briefly, pooled cryo-poor human plasma was fractionated according to theIgG purification scheme described herein, to provide a Fraction II+IIIprecipitate. The fraction II+III precipitate was re-suspended in lowconductivity extraction buffer (pH 5.1±0.2; ˜0.8 mS/cm) at a temperaturemaintained between 0° C. and 8° C. Aerosil® 380 (Evonik Industries AG)was added to a final concentration of between 40 and 60 g/kg II+IIIprecipitate. After the additional of CELPURE® C300 filter aid (AdvancedMinerals Corporation) to a final concentration of 0.5 kg/kg II+IIIprecipitate, the suspension was filtered using a depth filter. Theimmunoglobulin composition in the filtrate was then tested for FXIzymogen content. As shown in Table 22, treatment of the fraction II+IIIsuspension with finely divided SiO₂ resulted in nearly 90% reduction inthe Factor XI zymogen content of the composition.

TABLE 22 Impurities in the final FH012 composition. Fraction II + IIIRe-suspension Fraction II + III Extract Cuno Filtrate Fr. Fr. F-XI Fr.F-XI F-XI II + III II + III F-XI zymogen F-XI II + III F-XI zymogenzymogen paste, dissolved zymogen (1000s zymogen Filtrate volume, zymogen(1000s (% of II + III re- % Lot (kg) (L) (U/mL) of U) (%) (L) (U/mL) ofU) suspension) Removal 1 117 469 5.25 2460 100 2250 0.11 247 10.1% 89.9%2 118 475 5.13 2435 100 2290 0.11 251 10.3% 89.7% 3 119 479 4.51 2162100 2300 0.12 276 12.8% 87.2%

Example 4

To evaluate the elution of serine proteases from a finely divided SiO₂filter cake, as prepared in Example 3, elution buffers containingvarying concentrations of phosphate buffer (100, 50, 25, and 5 mM) wereused to elute proteins from the SiO₂ at two different pH (6.0, 7.5).Briefly, the filtercake was dissolved at a ratio of 1:5 in theappropriate buffer system and filtrated through depth filters (Cuno 50SA). The amidolytic activity and Factor H composition of each eluate wasthen determined (Table 23 and Table 24). As shown in Table 23, at lowerconductivity and pH (i.e., 6.0), the elution of amidolytic activitymeasured with the substrate CS2166 (FXIa, activated Protein C) wasreduced.

Under elution conditions at pH 7.5 (Table 24), Factor H elutiondecreased with increasing conductivity, while serine protease elutionincreased with increasing conductivity. Surprisingly, at extremely lowconductivity (5 mM phosphate; 0.882 mS/cm), serine protease elutionincreased substantially, while Factor H elution decreased. The dataobtained for elution at pH 7.5 is shown graphically in FIG. 3.

TABLE 23 Elution of Factor H and serine protease activity from finelydivided SiO₂ at pH 6.0. Substrate: CS2166 total Factor H Protein BufferSystem: pH = 6.0 Sample nmol*min [g/l Plasma] [nmol/g] 100 mM Phosphatebuffer; Filtrate 72745 0.27 61944 Cond. 11.88 mS/cm 50 mM Phosphatebuffer; Filtrate 65055 0.19 64600 Cond. 6.55 mS/cm 25 mM Phosphatebuffer; Filtrate 28591 0.05 63694 Cond. 3.48 mS/cm 5 mM Phosphatebuffer: Filtrate 4816 0.0003 57331 Cond. 0.882 mS/cm

TABLE 24 Elution of Factor H and serine protease activity from finelydivided SiO₂ at pH 7.5. Substrate: CS2166 total Factor H Protein BufferSystem: pH = 7.5 Sample nmol*min [g/l Plasma] [nmol/g] 100 mM Phosphatebuffer; Filtrate 236456 0.21 156718 Cond. 18.81 mS/cm 50 mM PhosphateBuffer; Filtrate 147829 0.29 109228 Cond. 10.91 mS/cm 25 mM Phosphatebuffer; Filtrate 84622 0.39 57892 Cond. 6.08 mS/cm 5 mM Phosphatebuffer: Filtrate 176685 0.33 134051 Cond. 1.524 mS/cm

Example 5

In order to demonstrate the ability to differentially elute serineproteases and Factor H co-bound to SiO₂, a two step elution procedurewas developed. Briefly, a fraction II+III filter cake formed after SiO₂treatment was prepared as before. The filter cake was then subjected toa first elution under solution conditions comprising an ionic strengthbetween 0.882 mS/cm and 11.88 mS/cm at pH 6.0. As demonstrated inExample 4, treatment of bound SiO₂ at low pH (pH 6.0) and low ionicstrength (less than 6.5 mS/cm) results in elution of serine proteases(e.g., FXIa), while a substantial fraction of Factor H remains bound.Subsequent treatment at high pH (pH 7.5) and high ionic strength resultsin the elution of Factor H from the SiO₂ (Table 25). Furthermore,consistent with the results provided in Example 4, initial treatment ofSiO₂ at high pH (7.5) results in elution of Factor H (Table 26). Asshown, an initial elution at lower conductivity and pH 6.0 could be usedto partially reduce amidolytic activity from the filter cake and thenFactor H can be eluted at 100 mM phosphate concentration, 150 mM NaCl,pH 7.6. This procedure resulted in a filtrate, Factor H yield of 0.31g/l plasma, with reduced amidolytic activity (CS2166) for furtherprocessing.

TABLE 25 Two-step differential elution of serine protease and Factor Hfrom SiO₂ at pH 6.0/7.6. First Elution buffer Second Elution Factor HSystem pH 6.0 buffer Sample [g/l Plasma] 100 mM Phosphate 100 mMPhosphate Filtrate 0.06 buffer; Cond. 11.88 buffer + 150 mM second mS/cmNaCl, pH 7.6 elution 50 mM Phosphate 100 mM Phosphate Filtrate 0.11buffer; Cond. 6.55 buffer + 150 mM second mS/cm NaCl, pH 7.6 elution 25mM Phosphate 100 mM Phosphate Filtrate 0.25 buffer; Cond. 3.48 buffer +150 mM second mS/cm NaCl, pH 7.6 elution 5 mM Phosphate 100 mM PhosphateFiltrate 0.31 buffer: Cond. 0.882 buffer + 150 mM second mS/cm NaCl, pH7.6 elution

TABLE 26 Two-step differential elution of serine protease and Factor Hfrom SiO₂ at pH 7.5/7.6. First Elution buffer Second Elution Factor HSystem pH 7.5 buffer Sample [g/l Plasma] 100 mM Phosphate 100 mMPhosphate Filtrate 0.05 buffer; Cond. 11.88 buffer + 150 mM second mS/cmNaCl, pH 7.6 elution 50 mM Phosphate 100 mM Phosphate Filtrate 0.06buffer; Cond. 6.55 buffer + 150 mM second mS/cm NaCl, pH 7.6 elution 25mM Phosphate 100 mM Phosphate Filtrate 0.06 buffer; Cond. 3.48 buffer +150 mM second mS/cm NaCl, pH 7.6 elution 5 mM Phosphate 100 mM PhosphateFiltrate 0.07 buffer: Cond. 0.882 buffer + 150 mM second mS/cm NaCl, pH7.6 elution

Example 6

To determine the amount of finely divided SiO₂ required for efficientremoval of serine proteases and serine protease zymogens from aplasma-derived protein composition, a fraction II+III precipitate (i.e.,II+III filtercake) was dissolved, filtered, treated with SiO₂, filteraid was admixed, and subjected to a second filtration. Briefly, thefraction II+III filtercake was first dissolved in 0.1 M phosphate buffercontaining 150 mM sodium chloride (pH 7.5; 30 mS/cm). This suspensionwas then filtered through a Cuno 50 SA filter and the filtratecollected. Aerosil 380 was admixed with the filtrate at a finalconcentration of either 1.0 or 2.5 g/g protein and then incubated for atleast 50 minutes. CELPURE filter aid was added and filtration wasperformed using a Cuno 50 SA filter. The resulting filtrate was thencharacterized for amidolytic activity, as reported in Table 27.Significantly, the results show that addition of Aerosil at a finalconcentration of 2.5 g/g protein reduced the amidolytic activity ofKallikrein, FXIa, and FXIIa in the composition by greater than 90%, ascompared to the sample treated with Aerosil at a final concentration of1.0 g/g protein.

TABLE 27 Amidolytic activity present in re-suspended Fraction II + IIIprecipitate after treatment with finely divided silicon dioxide.Reduction by increased Kallikrein, FXIa, FXIIa Substrate: S-2302 Aerosiladdition sample total: nmol*min [%] FH027 Cuno filtrate, 83347 — afteraddition of 1 g Aerosil per g Protein FH027 Cuno filtrate, 6227 92.5after addition of 2.5 g Aerosil per g Protein

Example 7

To evaluate the efficiency of SiO₂ treatment for the removal of FactorXI zymogen during the industrial-scale manufacture of plasma-derivedprotein compositions, the FXI zymogen content of six industrial-scalemanufacturing batches was characterized. Table 28 and Table 29 show theaverage FXI zymogen content of each upstream process step from threepurifications performed at the same manufacturing site. The data inTable 28 and Table 29 demonstrate that SiO₂ treatment ofmanufacturing-scale purifications can reduce the FXI zymogen content ofthe composition by at least 90%. Notably, manufacturing sites 1 admixedAerosil at a final concentration of 50 g/kg II+III precipitate, whilesite 2 used Aerosil at a final concentration of 40 g/kg II+IIIprecipitate. Surprisingly, this small difference in the amount ofaerosil used resulted in a significant difference in the Factor XIzymogen content of the filtrate after aerosil treatment (8.1% of Cohnstarting pool for site 2 vs. 2.8% of Cohn starting pool for site 1).

TABLE 28 Mean value of Factor XI zymogen content in each fraction ofthree large-scale manufacturing batches processed at site 1. F-XIzymogen Sample Volume (U/mL) (U) (% of Cohn pool) Cohn pool 3379 1.254233923 100.0 Supernatant I 3632 1.01 3669081 87.2 Supernatant II + II3927 0.21 812077 19.1 II + III paste* 2302 1.31 3026261 71.6 Filtrateafter Aerosil 2993 0.04 119107 2.8 Ppt G dissolved 248 0.31 77300 1.8

TABLE 29 Mean value of Factor XI zymogen content in each fraction ofthree large-scale manufacturing batches processed at site 2. F-XIzymogen Sample Volume (U/mL) (U) (% of Cohn pool) Cohn pool 2885 1.113193460 100.0 Supernatant I 3076 1.04 3208517 100.5 Supernatant II + II3376 0.29 968120 30.2 II + III paste* 474.3 4.96 2352714 74.0 Filtrateafter Aerosil 2280 0.11 258466.7 8.1 Ppt G dissolved 238.1 1.07253912.33 8.0

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method for preparing an Immunoglobulin G (IgG)composition having a reduced amount of a serine protease or a serineprotease zymogen, the method comprising the steps of: (a) precipitatinga cryo-poor plasma fraction, in a first precipitation step, with fromabout 6% to about 10% alcohol at a pH of from about 7.0 to 7.5 to obtaina first precipitate and a first supernatant; (b) precipitating IgG fromthe first supernatant, in a second precipitation step, with from about23% to about 27% alcohol at a pH of from about 6.7 to about 7.3 to forma second precipitate; (c) re-suspending the second precipitate to form asuspension; (d) contacting the suspension with finely divided silicondioxide (SiO₂) under a solution condition suitable to bind a serineprotease or serine protease zymogen; and (e) separating the SiO₂ fromthe suspension to form a clarified suspension.
 2. The method of claim 1,wherein step (b) comprises adjusting the ethanol concentration of thefirst supernatant formed in step (a) to about 25% (v/v) at a temperaturefrom about −7° C. to about −9° C.
 3. The method of claim 2, wherein step(d) comprises the addition SiO₂ to a final concentration of from about0.02 grams per gram precipitate formed in step (b) to about 0.06 gramsper gram precipitate formed in step (b).
 4. The method of claim 1,wherein the solution condition suitable to bind a serine protease orserine protease zymogen comprises a pH from 4.5 to 6.0 and aconductivity of from 0.1 mS/cm to 3 mS/cm.
 5. The method of claim 4,wherein the pH of the solution condition suitable to bind a serineprotease or serine protease zymogen is from 4.9 to 5.3.
 6. The method ofclaim 4, wherein the conductivity of the solution condition suitable tobind a serine protease or serine protease zymogen is from 0.5 mS/cm to 2mS/cm.
 7. The method of claim 1, wherein step (c) comprisesre-suspending the second precipitate formed in step (b) with are-suspension buffer containing phosphate and acetate, wherein the pH ofthe buffer is adjusted with from 300 mL to 700 mL of glacial acetic acidper 1000 L of re-suspension buffer.
 8. The method of claim 5, whereinthe conductivity of the solution condition suitable to bind a serineprotease or serine protease zymogen is from about 0.5 mS/cm to about 2mS/cm.
 9. The method of claim 1, wherein step (e) comprises thesub-steps of: (i) separating the SiO₂ from the suspension by filteringthe suspension through a filter press to form a clarified suspension;(ii) washing the filter press with at least 3 filter press dead volumesof a wash buffer having a pH of from about 4.6 to about 5.3, therebyforming a wash solution; and (iii) combining the clarified suspensionformed in sub-step (i) with the wash solution formed in sub-step (ii),thereby forming an enriched IgG composition.
 10. The method of claim 1,wherein the method further comprises the steps of: (f) precipitating IgGfrom the clarified suspension formed in step (e), in a thirdprecipitation step, with a final alcohol concentration of from about 22%to about 28% alcohol at a pH of from about 6.7 to about 7.3 to form athird precipitate; (g) re-suspending the third precipitate to form asecond suspension; and (h) separating the soluble fraction from thesecond suspension formed in step (e), thereby forming an enriched IgGcomposition.
 11. The method of claim 1, wherein the method furthercomprises an anion exchange chromatography enrichment step.
 12. Themethod of claim 1, wherein the method further comprises a cationexchange chromatography enrichment step.
 13. The method of claim 1,wherein the method further comprises at least one viral inactivation orremoval step.
 14. The method of claim 1, wherein the alcoholconcentration in the first precipitation step (a) is achieved by thespray addition of alcohol into the cryo-poor plasma fraction.
 15. Themethod of claim 1, wherein the alcohol concentration in the secondprecipitation step (b) is achieved by the spray addition of alcohol intothe first supernatant.
 16. The method of claim 10, wherein the alcoholconcentration in the third precipitation step (f) is achieved by thespray addition of alcohol into the clarified suspension.
 17. The methodof claim 1, wherein the pH of the first precipitation step (a) ismaintained throughout the first precipitation step by continuousmonitoring and adjustment of the pH.
 18. The method of claim 1, whereinthe pH of the second precipitation step (b) is maintained throughout thesecond precipitation step by continuous monitoring and adjustment of thepH.
 19. The method of claim 10, wherein the pH of the thirdprecipitation step (f) is maintained throughout the third precipitationstep by continuous monitoring and adjustment of the pH.
 20. The methodof claim 1, wherein step (b) comprises adjusting the ethanolconcentration of the first supernatant formed in step (a) to from about24% to about 26%.
 21. The method of claim 1, wherein step (b) isperformed at a temperature from about −7° C. to about −9° C.